RESEARCH ARTICLE

TPD52 expression increases neutral lipid storage within culturedcellsAlvin Kamili1,*, Nuruliza Roslan1,2,*, Sarah Frost1,2, Laurence C. Cantrill2,3, Dongwei Wang3,Austin Della-Franca1,2, Robert K. Bright4, Guy E. Groblewski5, Beate K. Straub6, Andrew J. Hoy7,Yuyan Chen1,2,‡ and Jennifer A. Byrne1,2,‡

ABSTRACTTumor protein D52 (TPD52) is amplified and/or overexpressed incancers of diverse cellular origins. Altered cellular metabolism(including lipogenesis) is a hallmark of cancer development, andprotein–protein associations between TPD52 and known regulatorsof lipid storage, and differential TPD52 expression in obese versusnon-obese adipose tissue, suggest that TPD52 might regulatecellular lipid metabolism. We found increased lipid droplet numbersin BALB/c 3T3 cell lines stably expressing TPD52, compared withcontrol and TPD52L1-expressing cell lines. TPD52-expressing 3T3cells showed increased fatty acid storage in triglyceride (from both denovo synthesis and uptake) and formed greater numbers of lipiddroplets upon oleic acid supplementation than control cells. TPD52colocalised with Golgi, but not endoplasmic reticulum (ER), markersand also showed partial colocalisation with lipid droplets coated withADRP (also known as PLIN2), with a proportion of TPD52 beingdetected in the lipid droplet fraction. Direct interactions betweenADRP and TPD52, but not TPD52L1, were demonstrated using theyeast two-hybrid system, with ADRP–TPD52 interactions confirmedusing GST pulldown assays. Our findings uncover a new isoform-specific role for TPD52 in promoting intracellular lipid storage, whichmight be relevant to TPD52 overexpression in cancer.

KEYWORDS: Lipid storage, Lipid droplet, Golgi, Tumor protein D52,PAT proteins

INTRODUCTIONTPD52 is a candidate oncogene located at chromosome 8q21.13,which is frequently amplified or gained in human cancer (Byrneet al., 2012, 2014; Shehata et al., 2008b). TPD52 overexpression hasbeen reproducibly associated with poor outcomes in breastcarcinoma (Byrne et al., 2014) and aggressive phenotypes in mostcancers examined (Adler et al., 2006; Bismar et al., 2006; Byrne

et al., 2014; Shehata et al., 2008b). TPD52 is the founding memberof the TPD52-like protein family, whose members share ∼50%sequence identity. At the molecular level, TPD52-like proteinsexhibit functional redundancy, in that heterologous partnersidentified through yeast two-hybrid screens using a singleTPD52-like bait also interact with related TPD52-like proteins(Wilson et al., 2001; Proux-Gillardeaux et al., 2003; Shahheydariet al., 2014). However, stable expression of TPD52 or its paralogueTPD52L1 in BALB/c 3T3 cells produced shared but also isoform-specific cellular effects (Lewis et al., 2007; Shehata et al., 2008a).Exogenous TPD52 but not TPD52L1 expression increase theproliferation and anchorage-independent growth of 3T3 cells,whereas both proteins produce similar morphological changes(Shehata et al., 2008a). Similarly, TPD52 but not TPD52L1transcript levels are significantly higher in breast carcinomasamples, relative to normal breast tissue (Shehata et al., 2008a).These results suggest that isoform-specific functions for TPD52 notshared by TPD52L1 underpin the oncogenic effects of TPD52overexpression.

A hallmark of cancer cells is deregulated cellular metabolism(Luo et al., 2009), with a number of studies focusing uponlipogenesis (Budhu et al., 2013; Kumar-Sinha et al., 2003; Wanget al., 2013). Actively proliferating cells require lipids to build newmembranes, lipid cofactors and lipid-modified proteins (Brasaemle,2007; Vander Heiden et al., 2009), yet the cytotoxicity of many lipidspecies requires their conversion into and storage as neutral lipids(e.g. triglycerides, TAG; cholesterol esters) within lipid droplets(Listenberger et al., 2003). Lipid droplets are highly complex,dynamic organelles that actively participate in lipid metabolismand cellular signalling, controlling intracellular lipid trafficking andinteracting with other organelles (Walther and Farese, 2012). Lipiddroplets consist of a neutral lipid core surrounded by a phospholipidmonolayer, and are coated by one or more members of the perilipin(PAT) family [perilipin, ADRP, TIP47 and S3-12 (also known asPLIN1–PLIN4, respectively), and OXPAT (also known as MLDPand PLIN5)] (Brasaemle, 2007) and a diverse array of other proteins(Krahmer et al., 2009; Walther and Farese, 2012). It is commonlyproposed that lipid droplets form within the endoplasmic reticulum(ER) and are transported from the ER to the Golgi, where more TAGis loaded and more proteins are attached (Fujimoto and Parton,2011; Walther and Farese, 2012; Wilfling et al., 2014).

Lipid droplets are constitutively present in fat-storing cells,including adipocytes and steroidogenic cells. Although present inlow numbers in most other cell types, increased numbers of lipiddroplets can occur in cancer cells (Bozza and Viola, 2010). Alipogenic phenotype has been particularly associated with ERBB2-positive breast cancers. Increased fatty acid synthase (FASN)expression has been noted in response to exogenous ERBB2expression in breast cancer cells (Kumar-Sinha et al., 2003), andReceived 21 December 2014; Accepted 10 July 2015

1Molecular Oncology Laboratory, Children’s Cancer Research Unit, Kids ResearchInstitute, The Children’s Hospital at Westmead, Westmead, New South Wales 2145,Australia. 2Discipline of Paediatrics and Child Health, University of Sydney, TheChildren’s Hospital at Westmead, Westmead, New South Wales 2145, Australia.3Kids Research Institute Microscope Facility, The Children’s Hospital at Westmead,Westmead, New South Wales 2145, Australia. 4Department of Immunology andMolecular Microbiology and TTUHSC Cancer Center, Texas Tech University HealthSciences Center, Lubbock, TX 79430, USA. 5Department of Nutritional Sciences,University of Wisconsin, Madison, WI 53706, USA. 6Department of GeneralPathology, Institute of Pathology, Heidelberg 69120, Germany. 7Discipline ofPhysiology, School of Medical Sciences and Bosch Institute and Boden Institute ofObesity, Nutrition, Exercise and Eating Disorders, University of Sydney, Sydney,New South Wales 2006, Australia.*These authors contributed equally to this work

‡Authors for correspondence (yuyan.chen@health.nsw.gov.au;jennifer.byrne@health.nsw.gov.au)

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genes encoding other regulators of lipid metabolism might be co-amplified with ERBB2 at chromosome 17q (Kourtidis et al., 2010).TPD52 expression has been reproducibly associated with ERBB2expression in human breast cancer cell lines and tissues, and inmammary tissues from Erbb2 transgenic mice (Byrne et al., 2014;Kourtidis et al., 2010; Roslan et al., 2014). By contrast, knockdownof the Caenorhabditis elegans TPD52 orthologue F13E6.1significantly reduces lipid storage as assessed by a genome-widescreening study (Ashrafi et al., 2003), and expression microarrayanalyses have identified increases in TPD52 levels in mouse andhuman adipose tissue from obese versus lean subjects (Clementet al., 2004; Keller et al., 2008; Nadler et al., 2000). TPD52 was alsoidentified as a perilipin-binding partner in a yeast two-hybrid screen(Yamaguchi et al., 2006), and TPD52 co-immunoprecipitated withTIP47 and other proteins (Zhang et al., 2007). These studies suggestthe possible involvement of TPD52 in regulating lipid metabolism.Here, we show for the first time that TPD52 but not TPD52L1

expression increases lipid droplet numbers in cultured cells, and thatTPD52 expression also promotes fatty acid storage in TAG.We alsodemonstrate that TPD52 colocalised with both Golgi markers andADRP-coated lipid droplets, with further evidence supporting adirect interaction between TPD52 and ADRP.

RESULTSStable TPD52 expression increased cellular lipid dropletnumbers in 3T3 fibroblast and MDA-MB-231 breast cancercellsWe have previously reported the effects of TPD52 and TPD52L1expression in 3T3 fibroblast cells (Shehata et al., 2008a), whosesequences alignments are shown in Fig. 1A. In the present study, weassessed lipid droplets in BALB/c 3T3 parental cells and those stablytransfected with PG307 vector only (vector-2 and/or vector-3),PG307-TPD52 (cell lines denoted D52-1-12, D52-2-1 andD52-2-7), or PG307-TPD52L1 (cell lines denoted D52L1-4 andD52-L1-6), using BODIPY 493/503 staining. Compared with parentand vector (vector-3) cells, TPD52-expressing cell lines (D52-2-1,D52-2-7) showed strikingly increased cytosolic lipid dropletnumbers (Fig. 1B; data not shown). TPD52L1-expressing 3T3 celllines showed similar lipid droplet staining to parental and vector-3control cell lines (Fig. 1B). Quantification of lipid dropletsconfirmed a significant increase in mean lipid droplet numbers percell in two TPD52-expressing 3T3 cell lines, but not in twoTPD52L1-expressing 3T3 cell lines, compared to vector control cells(Fig. 1C). This corresponded to an∼10- and 4-fold increase in meanlipid droplet numbers per cell in D52-2-7 cells and D52-2-1 cells,respectively, relative to vector-3 control cells (Fig. 1C). There wasalso a smaller but significant increase in the mean lipid droplet area(μm2) in D52-2-7 cells compared with vector-3 control cells(Fig. 1D). Levels of TPD52 or TPD52L1 in respective cell lineswere validated using western blot analyses (Fig. 1E). Electronmicroscopy analyses of vector-3 andD52-2-7 cells further confirmedlipid droplet accumulation in D52-2-7 cells (supplementary materialFig. S1A,B). Numerous whorled and laminated structures were alsonoted in D52-2-7 cells (supplementary material Fig. S1B), whichresembled phospholipid inclusions (O’Farrell et al., 2001).To determine whether these phenotypes were confined to 3T3

cells, we examined a second stable TPD52-expressing cell line,derived from the breast cancer cell line MDA-MB-231 with lowendogenous TPD52 expression levels (Roslan et al., 2014). Again,increased lipid droplets were detected in TPD52-transfected MDA-MB-231 cells (TPD52-H1D2) compared to vector control cells(supplementary material Fig. S1C).

TPD52-expressing 3T3 cells form more lipid dropletsfollowing oleic acid supplementationWe next examined how 3T3 cell lines would respond to exogenousfatty acid supplementation. Following 400 µM oleic acid treatmentfor 24 h, lipid droplet numbers per cell were significantly increasedin all 3T3 cell lines examined, relative to parallel cultures harvestedprior to oleic acid supplementation (Fig. 2A,B). However,significantly more lipid droplets per cell were measured inTPD52-expressing cells, compared with parent and vector controlcells (Fig. 2A,B). Quantification of lipid droplet numbers showeda significant increase (∼2-fold) in the mean lipid droplet numberper cell in all three TPD52-expressing cell lines post oleic acidtreatment, compared with oleic-acid-treated vector control cells(Fig. 2B). All three TPD52-expressing 3T3 cells showed verysimilar mean lipid droplet numbers per cell after oleic acidtreatment, despite different basal numbers of lipid droplets percell (Fig. 2B). Individually, there were ∼18- and ∼14-fold increasesin mean lipid droplet numbers per cell in parent and vector controlcells, respectively, after oleic acid treatment, but only ∼4-foldincreases in D52-1-12 and D52-2-1 cells, and a 2-fold increase inD52-2-7 cells (Fig. 2C). Increased lipid droplet numbers after 24 holeic acid treatment were associated with significantly increasedlipid droplet areas in each cell line (Fig. 2D), with similar relativefold changes in lipid droplet areas measured post oleic acidtreatment in all 3T3 cell lines analysed (Fig. 2E). However,significantly larger lipid droplets were measured in two of the threeTPD52-expressing cell lines (D52-1-12 and D52-2-7) post oleicacid treatment, compared with oleic-acid-treated vector control cells(Fig. 2D).

TPD52 expression alters fatty acid metabolism in 3T3fibroblast cellsTo explore the mechanisms by which TPD52 increases cellular lipidstorage, we compared fatty acid metabolism in 3T3 cell lines.Consistent with significant increases in lipid droplet numbers, thelevels of cellular TAG, the main component of lipid droplets, werealso significantly elevated in TPD52-expressing cells, most notablyin D52-2-7 cells, where TAG levels were approximately four timeshigher than those of vector controls (Fig. 3A). Cells were treatedwith[3H]acetic acid, to trace fatty acid synthesis, and with [1-14C]oleate,which can be taken up by cells. The rates of de novo fatty acidincorporation into TAG (de novo lipogenesis) were significantlyincreased in D52-1-12 (by ∼2-fold) and D52-2-7 (by ∼1.5-fold)cells compared with parent and vector cells (Fig. 3B). Similarly, theincorporation rates of medium-supplied [1-14C]oleate into TAGwere also significantly increased in D52-1-12 (∼2-fold) and D52-2-7(∼1.5-fold) cells (Fig. 3C). In contrast, rates of [1-14C]oleate uptakediffered significantly between vector control and parent 3T3 cells,but not between vector control and D52-2-7 cells (Fig. 3D), despitethe latter consistently demonstrating the highest number of lipiddroplets per cell (Fig. 1B,C; Fig. 2A,B).

To examine the molecular mechanisms contributing to increasedfatty acid incorporation into TAG observed in TPD52-expressingcells, we examined the levels of key enzymes in 3T3 cell linescultured without oleic acid supplementation, by using western blotanalyses. There were no significant differences in the levels ofFASN (Fig. 3E), acetyl-CoA carboxylase (Acc) (data not shown),stearoyl-CoA desaturase 1 (Scd1) (Fig. 3E) or diacylglycerolacyltransferase 2 (DGAT2) (data not shown), according to TPD52expression status. Similarly, there were no obvious differences inADRP levels, the major PAT protein that regulates lipid droplets innon-adipocytes (Brasaemle et al., 1997) (Fig. 3E), and no perilipin

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Fig. 1. Increased lipid storage in TPD52- but not TPD52L1-expressing 3T3 cells. (A) Alignment of TPD52 (Uniprot identifier P55327-2) and TPD52L1 (Uniprotidentifier Q16890-1) sequences, shown using the one-letter code, produced by the EMBOSSNeedle algorithm. Numbers to the left and right of sequences refer toamino acid positions. Vertical lines and colons indicate identical and conserved residues, respectively, whereas hyphens represent gaps inserted to producethe alignment. PEST domains (red text) (Byrne et al., 1996), coiled-coil domains (underlined text) (Byrne et al., 1998), the TPD52 Ser136 phosphorylation site(bold text) (Thomas et al., 2010), and the TPD52L1 consensus 14-3-3-binding site (bold text) (Boutros et al., 2003) are indicated by arrows. A schematic diagramof the exon and domain composition of TPD52 isoforms has also been reported by Byrne et al. (2014). (B) Immunofluorescence analyses of control (3T3-parent,vector-3), TPD52-expressing (D52-2-1, D52-2-7) and TPD52L1-expressing (D52L1-4, D52L1-6) 3T3 cells, stained with BODIPY 493/503 (green) for lipiddroplets and DAPI (blue) for nuclei. Images are representative of those obtained in three independent experiments. (C) Quantification of lipid droplet numbers percell using Image-Pro Plus Version 5.1 software. Images of at least ten panels per cell line in each of three independent experiments were quantified, and datarepresent means±s.e.m. (D) Quantification of lipid droplet area (μm2) as described in C. P-values in C and D were calculated with a Student’s t-test. (E) Westernblot analyses of 3T3 cells with antisera to the proteins shown (left). Gapdh served as a loading control. MW, molecular mass (right). Results are representative ofthree independent experiments.

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Fig. 2. TPD52-expressing cells storemore lipids upon oleic acid supplementation. (A) Indirect immunofluorescence analyses of control (3T3-parent, vector-3), and TPD52-expressing (D52-1-12, D52-2-1, D52-2-7) 3T3 cells with (right panels, 24 h) or without (left panels, No) 400 μM oleic acid in fatty-acid-free BSA(OA). An enlarged image of a D52-2-7 cell post-supplementation (white boxed region) is shown. Lipid droplets were stained with BODIPY 493/503 (green) andnuclei were stained with DAPI (blue). Images shown are representative of those obtained in three independent experiments. (B) Quantification of lipid dropletnumbers per cell using Image-Pro Plus 5.1 software as described in Fig. 1C. Data are presented as means±s.e.m. from three independent experiments. Lipiddroplet numbers were significantly increased in all cell lines post-oleic acid supplementation compared with no treatment (3T3-parent, P=5×10−5; vector-3,P=2×10−5; D52-1-12, P=1×10−5; D52-2-1, P=3×10−6; D52-2-7, P=0.002). Comparisons between vector-3 and TPD52-expressing cells post-oleic acid treatmentare also shown. (C) Relative fold changes in mean±s.e.m. lipid droplet numbers per cell (oleic acid-treated or untreated) for each cell type. (D) Quantification oflipid droplet area (μm2) as described in Fig. 1C. Lipid droplet areas were significantly increased in all cell lines post-oleic acid supplementation compared withno treatment (3T3-parent, P=0.05; Vector-3, P=1×10−4; D52-1-12, P=2×10−6; D52-2-1, P=0.004; D52-2-7, P=4×10−4). Comparisons between vector-3 andTPD52-expressing cells post-oleic acid treatment are also shown. NS, not statistically significant, P>0.05. (E) Relative fold changes in mean lipid droplet area.All P-values were calculated with a Student’s t-test.

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Fig. 3. Altered fatty acid metabolism in TPD52-expressing 3T3 cells. (A) Triglyceride levels measured in parent, vector and TPD52-expressing 3T3 cells.Incorporation rates of (B) newly-synthesised free fatty acid (from [3H]acetate) into TAG, as a measure of de novo lipogenesis and (C) [1-14C]oleate into TAG,and (D) [1-14C]oleate uptake rates were measured in 3T3-parent, vector control, and TPD52-expressing 3T3 cells following 4 h incubation with [3H]acetic acid(1 µC/ml), [1-14C]oleic acid (0.5 µC/ml) and 0.5 mM unlabelled oleate. For A–D, three independent experiments were performed and differences between vectorcontrols and TPD52-expressing cells were examined by Student’s t-test. NS, not statistically significant, P>0.05. The [1-14C]oleate uptake rate (D) was alsosignificantly increased in parent versus vector control cell lines (P=0.002, Student’s t-test). (E) Western blot analyses of 3T3 cells cultured without oleic acidsupplementation, with antisera to proteins shown (left). Gapdh served as a loading control. MW,molecular mass (right). Results are representative of at least threeindependent experiments. (F) MTT assays of parent, vector controls and TPD52-expressing 3T3 cells treated with 250 μM palmitic acid for 4 days. Results arepresented as percentages of MTT absorbance at indicated time points relative to that at day 0. Data are means±s.e.m. from three independent experiments.Significant differences (Student’s t-test) were detected between D52-1-12 and vector controls (Day 1, P=0.03; Day 2, P=0.005; Day 3, P=0.01; Day 4, P=0.004),and D52-2-7 and vector controls (Day 1, P=0.01; Day 2, P=0.003; Day 3, P=0.001; Day 4, P=0.004).

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was detected as a marker of adipocyte differentiation (data notshown).Excess free saturated fatty acids such as palmitic acid are toxic to

cells, whereas unsaturated fatty acids can rescue palmitate-inducedapoptosis by channelling palmitate into triglyceride pools and awayfrom pathways leading to apoptosis (Listenberger et al., 2003). Weinvestigated whether TPD52 expression would provide protectiveeffects to cells upon palmitic acid treatment. MTT assays performedover 4 days post 250 μM palmitic acid treatment showedsignificantly increased cell survival in D52-1-12 and D52-2-7cells, compared with that of parent and vector cells (Fig. 3F).

Colocalisation of TPD52 with a Golgi but not an ERmarker inTPD52-expressing cellsStudies have indicated that lipid droplet formation is initiated inthe ER (Martin and Parton, 2006). Indirect immunofluorescenceanalyses of TPD52 show granular cytoplasmic staining with aperinuclear accentuation in MCF-7 breast cancer (Balleine et al.,2000) and TPD52-expressing 3T3 cells (Chen et al., 2013). AsTPD52 further accumulated at the perinuclear region of TPD52-expressing 3T3 cells upon oleic acid treatment (supplementarymaterial Fig. S2B), we examined whether TPD52 could beassociated with the ER by staining for protein disulfide isomerase(Pdi) (Ohsaki et al., 2008). Although Pdi staining was detectedthroughout the cytoplasm in both D52-2-7 and vector control cells,with or without 24 h of 400 µM oleic acid treatment (supplementarymaterial Fig. S2), Pdi did not obviously colocalise with TPD52 inD52-2-7 cells (supplementary material Fig. S2B).The Golgi complex also plays an important role in lipid droplet

growth (Hesse et al., 2013; Kalantari et al., 2010), so we comparedTPD52 staining with that of the Golgi matrix protein Gm130(Golgin 95, also known as GOLGA2) (Marra et al., 2001). Althoughthere were no obvious differences in Gm130 staining betweenD52-2-7 (Fig. 4A) and vector control cells (supplementary materialFig. S3A,B), perinuclear TPD52 staining colocalised with Gm130in D52-2-7 cells (Fig. 4A). Following 6 h of 400 µM oleic acidtreatment, this colocalisation became more prominent in some cells(Fig. 4B, cells 1 and 2). Quantification of the extent of colocalisationbetween Gm130 and TPD52 (Fig. 4) using Pearson’s correlationand Manders’ overlap coefficients showed that this significantlyincreased from median values of 0.31 and 0.42 (no oleic acid, n=28)to 0.38 and 0.48 (6 h oleic acid, n=23) (Pearson’s correlationcoefficients, P<0.0001; Manders’ overlap coefficients, P<0.0001;Mann–WhitneyU-test, data not shown). Colocalisation of TPD52 andGm130 was further confirmed in D52-1-12 and D52-2-1 cells withand without oleic acid treatment (supplementary material Fig. S4).

Colocalisation of TPD52 with ARL1 on Golgi in TPD52-expressing cellsThe GTPase ADP-ribosylation factor-like 1 (ARL1) coordinatesrecruitment of Golgin proteins from the cytosol to Golgi membranesin a GTP-dependent manner (Gillingham and Munro, 2003), whichalso requires ADP-ribosylation factor-related protein 1 (ARFRP1)(Zahn et al., 2006). It has been proposed that the ARFRP1–ARL1–Golgin–Rab cascade participates in the control of lipid dropletformation (Hesse et al., 2013; Hommel et al., 2010).We investigatedwhether ARL1 colocalised with Gm130 as well as TPD52. Asexpected (Lu et al., 2004), ARL1 showed incomplete peri-nuclearcolocalisation with Gm130 in D52-2-7 cells with or without oleicacid treatment (Fig. 5A), and in vector control cells (supplementarymaterial Fig. S3). ARL1 also colocalised with TPD52 in untreatedD52-2-7 cells, with more prominent colocalisation detected after

oleic acid treatment (Fig. 5B), and comparable results were obtainedin D52-1-12 and D52-2-1 cells (data not shown). As for Gm130,quantification of the extent of colocalisation between ARL1 andTPD52 (Fig. 5B) using Pearson’s correlation and Manders’ overlapcoefficients showed that this significantly increased from medianvalues of 0.31 and 0.59 (no oleic acid, n=21) to 0.41 and 0.66 (24 holeic acid, n=21) (Pearson’s correlation coefficients, P=0.0022;Manders’ overlap coefficients, P=0.0003; Mann–Whitney U-test,data not shown). As the fixation and permeabilisation methodrequired for ARL1 detection did not allow for BODIPY 493/503 co-staining, we used ADRP as a surrogate for lipid droplet staining(Imamura et al., 2002), having also confirmed the co-incidence ofADRP and BODIPY 493/503 staining in TPD52-expressing 3T3cells (data not shown). Western blot analyses showed no differencesin ARL1 or ARFRP1 levels between TPD52-expressing and controlcells (supplementary material Fig. S3C).

Limited colocalisation of TPD52 and ADRP in TPD52-expressing 3T3 cellsADRP is the predominant PAT protein that coats small lipid dropletsin non-adipocytes (Bickel et al., 2009). We therefore compared thesubcellular distribution of ADRP and TPD52 in 3T3 cell lines.ADRP showed clustered ring structures in D52-2-7 cells, whichwere largely excluded from the Golgi and TPD52 perinuclear region(Fig. 5; Fig. 6A,B, cell 1). However, a proportion of TPD52 wasdetected on ADRP-stained ring structures resembling lipid dropletsin both untreated (Fig. 5B; Fig. 6A) and oleic acid-supplementedD52-2-7 cells, with the latter showing larger grape-like ADRP-stained clusters (Fig. 5B; Fig. 6B, cell 2), than those detected invector control cells (supplementary material Fig. S3A,B; data notshown). Quantification of the extent of colocalisation betweenADRP and TPD52 (Fig. 6) using Pearson’s correlation andManders’ overlap coefficients showed that this significantlyincreased from median values of 0.15 and 0.17 (no oleic acid,n=25) to 0.29 and 0.33 (6 h oleic acid, n=19) (Pearson’s correlationcoefficients, P<0.0001; overlap coefficients, P<0.0001; Mann–Whitney U-test; data not shown).

Altered lipid droplet distribution in TPD52-expressing versusvector control 3T3 cellsWe also noted differences in the distribution of ADRP-stained lipiddroplets between TPD52-expressing and vector control cells.ADRP-stained lipid droplets formed clusters towards theperiphery of D52-2-7 cells, which became more prominent uponoleic acid treatment (Fig. 6; Fig. 7A), whereas lipid dropletsaccumulated closer to the nuclei of vector-2 cells (Fig. 7A;supplementary material Fig. S3A,B). To further address this, wequantified the distance between the centre of lipid droplets to thecentre of the respective cell nucleus, with more than 1900 lipiddroplets quantified for each setting. In vector control cells, 96.6%ADRP-stained bodies were distributed less than 100 pixels from thecentre of each nucleus (Fig. 7B). In contrast, 83.7% objects in D52-2-7 cells were distributed less than 100 pixels from the centre ofeach nucleus, with oleic acid treatment having no significant effecton either distribution (Fig. 7B). Statistical analyses confirmedsignificant differences in lipid droplet distributions between D52-2-7 and vector cells with and without oleic acid treatment (Fig. 7C).

Direct interactions between TPD52, but not TPD52L1, andADRP or TIP47Based on the colocalisation of TPD52 andADRP, and possible rolesfor TPD52 in lipid droplet formation or trafficking, we investigated

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whether TPD52 (Fig. 1A) directly interacts with ADRP. We firstemployed the yeast two-hybrid system which has previously beenused to identify TPD52-binding partners (Boutros et al., 2003;Byrne et al., 1998; Proux-Gillardeaux et al., 2003; Shahheydariet al., 2014). Co-transfection of amino acids 1–415 ADRP or full-length TIP47 bait and TPD52 prey constructs produced detectableHf7c yeast growth on triple drop-out medium after 5 days and3 days, respectively (Fig. 8A). However, co-transfection of TIP47 or

ADRP bait with TPD52L1 prey (Fig. 1A) resulted in no detectableyeast growth after 9 days (Fig. 8A).

To further confirm interactions between TPD52 and ADRP, weperformed GST pulldown assays. Given that the molecular mass ofADRP and GST-tagged TPD52 are both ∼50 kDa, GST proteinswere cross-linked to GSH beads. ADRP was recovered by GST-tagged mouse and human TPD52, but not by the GST tag alone,from D52-1-12 and D52-2-7 3T3 cell lysates (Fig. 8B). Interactions

Fig. 4. Colocalisation of TPD52 and the Golgimarker Gm130 in untreated and oleic-acid-treated D52-2-7 cells. Cells were untreated(A) or supplemented with 400 μM oleic acid infatty-acid-free BSA (OA) for 6 h (B), fixed andsubjected to indirect immunofluorescenceanalyses. Cells were colabelled with TPD52(red), and Gm130 (green). Enlarged images ofthe regions indicated by white boxes are shownbelow, with colocalisation shown in yellow in themerge image. Images shown are representativeof those obtained in at least three independentexperiments.

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between GST-tagged TPD52 and Rab5 were used as positivecontrols (Shahheydari et al., 2014) (Fig. 8B).To examinewhether TPD52 also localises in lipid droplet fractions,

we isolated lipid droplet fractions from D52-2-7 cells treated with400 μM oleic acid for 24 h using sucrose density centrifugation. As

previously reported (Brasaemle and Wolins, 2006), lipid dropletsfloated to the top of the gradient and were enriched in ADRP(Fig. 8C). A small proportion of both TPD52 and ARL1 weredetected in this fraction, as was Rab5 (Brasaemle et al., 2004; Choet al., 2007; Liu et al., 2007; Sato et al., 2006) (Fig. 8C). However, we

Fig. 5. Colocalisation of Gm130 or TPD52 with ARL1 and ADRP in untreated and oleic-acid-treated D52-2-7 cells. Cells were untreated or supplementedwith 400 μM in fatty-acid-free BSA (OA) for 24 h, fixed, and subjected to indirect immunofluorescence analyses. Cells were colabelled with either (A) Gm130(green) or (B) TPD52 (green), ARL1 (red), and ADRP (pseudo-coloured, red), with colocalisation shown in yellow in the merge image. Images shown arerepresentative of those obtained in three independent experiments.

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did not detect Snap23 or Vamp4 within this lipid droplet fraction(Fig. 8C), even after longer exposures (data not shown). This contrastswith the findings of Boström et al. (2007), who reported that Snap23and Vamp4, as well as NSF, α-Snap and Syntaxin-5, were associatedwith lipid droplets in ADRP-transfected NIH-3T3 cells. Weconfirmed no obvious changes in the protein levels of Nsf1, Snap

23, syntaxin-5 and syntaxin-6 and Vamp4 according to TPD52expression status in 3T3 cells (Fig. 8D).

DISCUSSIONDespite increasing recognition of TPD52 overexpression in variouscancer types (Tennstedt et al., 2014; Byrne et al., 2014), little is

Fig. 6. Colocalisation of ADRP and TPD52 in untreated and oleic-acid-treated D52-2-7 cells. Cells were prepared as described in Fig. 4. Cells werecolabelled with TPD52 (green) and ADRP (pseudo-coloured, red). Enlarged images of the regions indicated by white boxes are shown below, with colocalisationshown in yellow. Images shown are representative of those obtained in three independent experiments.

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known about the molecular or cellular functions of TPD52. Thisstudy has provided experimental evidence that TPD52 regulatescellular lipogenesis and lipid storage, which supports previous

results of high-throughput studies showing, for example, thatF13E6.1 (TPD52) knockdown in C. elegans significantly reduceslipid storage (Ashrafi et al., 2003), and that there are increases in

Fig. 7. Altered distributions of lipid droplets in untreated and oleic-acid-treated D52-2-7 cells versus vector control cells. (A) Representative imagesof Vector-2 and D52-2-7 cells with or without 400 μM oleic acid (OA) treatment. ADRP (pseudo-coloured, green), DAPI (blue). (B) Quantification of distances(pixels) between the centre of each lipid droplet object to the centre of the respective cell nucleus. At least 20 cells per cell type per treatment were assessed usingimages randomly chosen from three experiments, and frequencies (y-axis) of distances (x-axis) were plotted on histograms. (C) Statistical analyses confirmedsignificant differences in lipid droplet distributions between D52-2-7 and vector cells with or without oleic acid treatment (Mann–Whitney U-test). Boxes extendfrom the 25th percentile to the 75th percentile, with horizontal lines indicating median values. The whiskers show the range. Graphs were generated usingGraphPad Prism 4.

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Fig. 8. Direct interactions between TPD52 andADRPor TIP47. (A) Analyses of direct interactions between TPD52 or TPD52L1 and ADRPor TIP47, along withknown positive and negative controls using the yeast two-hybrid system. (+), growth on solid SD/−His−Leu−Trp medium at 30°C after 5–6 days; (++), after3–4 days; (+++), or after 1–2 days; (−), no visible growth after at least 7 days; ND, not determined. Results were obtained from three independent experimentswhere all interactions were tested in triplicate. (B) GST pulldown assays using GST-tagged full-length mouse Tpd52 or human TPD52 or GST tag, and D52-1-12(left) or D52-2-7 (right) 3T3 cell lysates. Upper and middle panels show results of western blot analyses using anti-ADRP antibody and anti-Rab5 antisera,respectively. The lower panel shows Ponceau S staining to reveal mouse and human GST–Tpd52 and GST–TPD52, respectively, or the GST tag before cross-linking (arrows). At least three independent experiments were performed. (C)Western blot analyses [proteins detected are indicated on the left, molecularmasses(MW) are indicated on the right] using lipid droplet (LD) fractions from D52-2-7 cells treated with 400 μMoleic acid in fatty-acid-free BSA for 24 h. Proteins detectedin the lipid droplet fraction were compared with those detected in total protein from identically treatedD52-2-7 cells. Gapdh served as a negative control. Results arerepresentative of three independent experiments. (D) Detection of SNARE protein levels according to TPD52 expression status in 3T3 cell lines. Total proteins wereextracted from 3T3 cells and subjected towestern blot analyses. Antisera to the proteins examined are shown on the left, andmolecularmass (MW) is shown on theright. α-Tubulin served as a loading control. Results are representative of three independent experiments.

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Tpd52 or TPD52 levels in mouse and human adipose tissue fromobese versus lean subjects (Clement et al., 2004; Keller et al., 2008;Nadler et al., 2000). Our findings are also consistent with TPD52being androgen-inducible in prostate cancer (Rubin et al., 2004;Wang et al., 2004), with androgen representing a potent regulator oflipogenesis and lipid metabolism in this disease (Massie et al., 2011;Swinnen and Verhoeven, 1998).Our results suggest that TPD52 enhances lipid storage through

more than one mechanism. Increased numbers of lipid droplets wereconsistently noted in TPD52-expressing 3T3 fibroblastic cell linesand MDA-MB-231 breast cancer cells with exogenously increasedTPD52 levels, relative to vector controls. Following oleic acidsupplementation, all three TPD52-expressing 3T3 cell lines alsoshowed significantly increased lipid droplet numbers comparedwith both untreated cells and oleic acid-treated vector control cellswithout corresponding differences in fatty acid uptake rates, whichcould underpin these phenotypes. This suggests that TPD52contributes to lipid droplet formation within the ER, which issupported by previous reports of TPD52 being an ER-associatedprotein (Hoja et al., 2000). However, TPD52 showed no detectablecolocalisation with Pdi in TPD52-expressing 3T3 cells, but insteadshowed perinuclear colocalisation with the Golgi markers Gm130andARL1, in the presence and absence of oleic acid supplementation.TPD52, Gm130 and ARL1 detection became more prominentwithin this perinuclear compartment following oleic acid treatment,as did the degree of colocalisation between TPD52 and Gm130 orARL1 in some cells. This suggests that rather than influencinglipid droplet formation within the ER, TPD52 might contribute tolipid loading within the Golgi, leading to growth of lipid dropletswithin this compartment and facilitating their detection by lightmicroscopy. Expansion of the Golgi compartment and increasedcolocalisation between TPD52 and Gm130 and ARL1 in oleic-acid-supplemented 3T3 cell lines indicates that these cells increase theirreliance upon Golgi function in response to increased exogenouslipid loading. In support of a Golgi-located function for TPD52 inregulating lipid droplets, TPD52 has been reported to interact withmembers of two protein families involved in the ARFRP1–ARL1–Golgin–Rab cascade (Hesse et al., 2013), namely Rab and Golginproteins (Messenger et al., 2014; Shahheydari et al., 2014).In addition to localising to the Golgi complex in TPD52-

expressing 3T3 cell lines, TPD52 showed limited, more peripheral,colocalisation with the lipid droplet protein ADRP. A smallproportion of TPD52 was detected within the lipid dropletfraction of D52-2-7 cells, a finding that is supported by theDrosophila orthologue CG5174 having been previously detectedwithin the lipid droplet proteome (Cermelli et al., 2006). TPD52also bound both ADRP and TIP47 in a yeast two-hybrid system, andTPD52–ADRP interactions were validated using GST pulldowns.TPD52 was previously identified as a binding partner of perilipinresidues 1–240 in a yeast two-hybrid screen (Yamaguchi et al.,2006) and as a TIP47 co-immunoprecipitating protein (Zhang et al.,2007). This indicates that TPD52 might directly co-operate withPAT proteins, to promote either lipid droplet trafficking and/or lipidstorage at the lipid droplet surface.Our results identified increased lipid droplet numbers in TPD52-

but not TPD52L1-expressing 3T3 cell lines, and although wedetected interactions between TPD52 and ADRP or TIP47, similarinteractions were not detected for TPD52L1. At present, it is notknown why TPD52 but not TPD52L1 can bind PAT proteins.TPD52-like genes commonly include alternatively-spliced exonsencoding a 14-3-3-binding site (Boutros et al., 2003); the relevantexon is more commonly included in TPD52L1 transcripts (Nourse

et al., 1998) but less frequently included in TPD52 transcripts(Boutros et al., 2003; Nourse et al., 1998; Wang et al., 2004). A 14-3-3-binding site was present in the TPD52L1 isoform expressed in3T3 cells and employed in interaction testing, but was absent fromthe TPD52 isoform employed in these and other functional analyses(Boutros et al., 2003; Shehata et al., 2008a). It is possible thatthe presence of a 14-3-3-binding site in TPD52-like proteins actsas a molecular switch, allowing interactions with 14-3-3 proteinsand preventing interactions with PAT proteins. In support of thishypothesis, another TPD52-like protein TPD52L2 has beenreproducibly identified within lipid droplet fractions (Hodges andWu, 2010) from oleic-acid-treated A431 cells stably expressingstomatin (Umlauf et al., 2004), Hep39 cells (Sato et al., 2006), U937cells (Wan et al., 2007), differentiated 3T3-L1 cells (Cho et al.,2007) and 3T3-L1 cells cultured to stimulate lipolysis (Brasaemleet al., 2004). Increased TPD52L2 levels have also been reported inresponse to furan fatty acid treatment of Caco-2 cells (Lengler et al.,2012), and in the first reported mouse knockout model for anyTpd52-like gene, smaller body length and absent or minimal hepaticlipidosis were reported in Tpd52l2−/− females (Adissu et al., 2014).Like TPD52, TPD52L2 isoforms are predicted to lack 14-3-3-binding sites in most tissues (Boutros et al., 2003).

In addition to increased lipid droplet numbers that were measuredin all three TPD52-expressing 3T3 cell lines, two out of three ofthese cell lines (D52-1-12 and D52-2-7, the latter harbouring thehighest TPD52 levels and lipid droplet numbers) also showedsignificantly increased fatty acid incorporation into TAG from bothde novo fatty acid synthesis, and fatty acid uptake. Significantlylarger lipid droplets were also noted in D52-1-12 and D52-2-7 cellswhen they were supplemented with oleic acid, and in D52-2-7 cellsin the absence of oleic acid treatment. This indicates that in additionto mechanisms regulating lipid droplet numbers, TPD52 can eitherdirectly or indirectly alter other aspects of lipid metabolism withincells, although TPD52 expression might not regulate fatty aciduptake rates. The functional significance of this phenotype wasdemonstrated by D52-1-12 and D52-2-7 cells also showingimproved cell survival in response to palmitic acid supplementation.

Lipids play diverse roles in building cellular structures, by formingmembrane microdomains for functional scaffolding of proteincomplexes, serving as fat storage depots and acting as signallingmolecules (Baumann et al., 2013; Currie et al., 2013; Nomura andCravatt, 2013; Zadra et al., 2013). All of these processes are importantin cancer and, as such, altered lipid metabolism is now an establishedhallmark of cancer (Baumann et al., 2013; Currie et al., 2013; NomuraandCravatt, 2013; Zadra et al., 2013). Both breast and prostate cancersshow elevated levels of intracellular lipid (Fisher et al., 1977; Swinnenand Verhoeven, 1998), and are characterised by frequent TPD52overexpression (Roslan et al., 2014; Rubin et al., 2004; Shehata et al.,2008a; Wang et al., 2004), as well as TPD52 amplification inclinically important disease subsets (Cornen et al., 2014; Guedj et al.,2012; Liu et al., 2013). As androgen upregulates lipogenesis inprostate cancer cells (Swinnen and Verhoeven, 1998; Massie et al.,2011), our results suggest that androgen-induced TPD52 upregulationmight contribute to this important process. ERBB2-amplified breastcancers also show increased lipogenesis because ERBB2 both directlyand indirectly regulates FASN function (Kumar-Sinha et al., 2003;Menendez and Lupu, 2007), and the co-amplification of otherchromosome 17q genes encoding regulators of lipid metabolism(Kourtidis et al., 2010). As TPD52 is co-expressed with ERBB2 inhuman breast cancer cell lines and tissues (Byrne et al., 2014;Kourtidis et al., 2010; Roslan et al., 2014), and is a known survivalfactor in ERBB2-amplified breast cancer cell lines (Kourtidis et al.,

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2010; Roslan et al., 2014), elevated TPD52 expressionmight similarlyadvantage ERBB2-amplified breast cancer cells by increasing bothlipogenesis and lipid storage capacity. The isoform-specificity of thesefunctions might partly explain why TPD52 but not TPD52L1 istargeted by gene amplification in cancer.In summary, this study has identified a new isoform-specific role

for TPD52 as a promoter of both lipid storage and lipogenesis thatmight contribute to an improved understanding of lipid dropletfunction under both physiological and pathological conditions.Given previous reports of increased TPD52 or Tpd52 transcriptlevels in adipose tissue from obese versus non-obese subjects(Clement et al., 2004; Keller et al., 2008; Nadler et al., 2000),inhibition of TPD52 function might have future relevance in themanagement of obesity. As TPD52 amplification andoverexpression is also frequent in lipogenic cancers such as breastand prostate cancer (Roslan et al., 2014; Rubin et al., 2004; Shehataet al., 2008a; Wang et al., 2004), our results might also partiallyexplain TPD52 targeting in these diseases, and could similarly beapplied to target lipogenic cancers in future.

MATERIALS AND METHODSCell lines and cell cultureHuman breast carcinoma cells (MDA-MB-231) were cultured in GIBCO®

RPMI 1640 (Life Technologies, VIC, Australia) medium supplementedwith 10% fetal bovine serum (FBS; Life Technologies), 6 mM L-glutamine(Life Technologies) in a humidified atmosphere containing 5% CO2 at37°C. Cell line identities were confirmed through short tandem repeatprofiling by CellBank Australia (Westmead, NSW, Australia). Vector-,TPD52- and TPD52L1-transfected BALB/c 3T3 cell lines are as previouslyreported (Shehata et al., 2008a).

Derivation of stably TPD52-expressing MDA-MB-231 cell linesThe PG307-TPD52 plasmid was constructed by subcloning a SalI–BamHITPD52 fragment (GenBank NM_005079.3, encoding the TPD52 isoformshown in Fig. 1A), representing the 91-bp 5′UTR, 555-bp coding sequenceand 2599-bp 3′UTR, into the PG307 expression vector (Boutros et al.,2003). After verification of constructs using Sanger sequencing, the PG307-TPD52 construct or PG307 vector alone were stably transfected into MDA-MB-231 cells, as described previously (Boutros et al., 2003).

Indirect immunofluorescence analysesCells were plated onto glass coverslips in six-well plates, and culturedovernight to reach 70–80% confluence the next day. For lipid dropletquantification, untreated cells or cells supplemented with 400 µM oleic acid(Sigma-Aldrich, NSW, Australia) complexed to 10% fatty-acid-free BSA(Sigma-Aldrich) (Listenberger and Brown, 2007) for 24 h were fixedin 3% formaldehyde in PBS for 30 min at room temperature, then stainedwith 1 µg/ml BODIPY 493/503 (Life Technologies) for 30 min atroom temperature. DNA was counterstained using 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI; Sigma-Aldrich).

For co-staining of lipid droplets and proteins, untreated cells or cellssupplemented with 400 µM oleic acid in fatty-acid-free BSA for 6 h were fixedwith 3% formaldehyde and 0.025% glutaraldehyde in PBS for 10 min at roomtemperature, treated with 50 mM ammonium chloride in PBS for 10 min,followed by permeabilisation using 0.1% Triton X-100 in PBS (Ohsaki et al.,2005). Cells were stained with affinity-purified rabbit polyclonal TPD52antisera (1:100) (Balleine et al., 2000) and GM130 mouse monoclonalantibody (1:1000, BD Transduction Laboratories, San Jose, CA), followed byincubation in 3% BSA in PBS containing 1 µg/ml BODIPY 493/503 solution,Cy3-conjugated anti-rabbit-IgG (Jackson ImmunoResearch Laboratories Inc,West Grove, PA), and Alexa-Fluor-633-conjugated anti-mouse-IgG (LifeTechnologies) secondary antibodies.

For other indirect immunofluorescence analyses, cells treated with orwithout 400 µM oleic acid were fixed in 3% formaldehyde in PBS for15 min at room temperature, permeabilised with cold acetone and methanol

(v/v, 1:1) for 15 min at −20°C, and then incubated with affinity-purifiedrabbit polyclonal TPD52 antisera and GM130 antibody as described above,or with antibodies against TPD52 (mouse monoclonal, 1:10; Tiacci et al.,2005), PDI (C81H6) rabbit monoclonal antibody (1:100, Cell SignalingTechnology, Danvers, MA), ARL1 (EPR10595) rabbit monoclonalantibody (1:100, Abcam, VIC, Australia), and guinea pig polyclonalantibody against ADRP (1:200, Progen Biotechnik, Heidelberg, Germany).This method was not compatible with BODIPY 493/503 staining, butallowedADRP detection at lipid droplets. Alexa-Fluor-488-conjugated anti-mouse-IgG (Life Technologies), Cy3-conjugated anti-rabbit-IgG (JacksonImmunoResearch Laboratories Inc), and Alexa-Fluor-633-conjugated anti-guinea-pig-IgG (Life Technologies) were utilised as secondary antibodies.All samples were viewed with a Leica LCS SP5 II confocal microscope(NSW, Australia) using a 63× objective lens.

Quantitative analysis of colocalisation between TPD52 and Gm130,ARL1 or ADRP with or without oleic acid supplementation was performedusing Image-Pro Analyzer 7.0 (MediaCybernetics, Rockville, MD).Scatterplots displaying intensity ranges of red and green pixels weregenerated from 19–28 representative single focal plane images for each casewithout pre-processing (TPD52, Gm130 and ARL1), or after backgroundsubtraction to obtain lipid droplet structures (ADRP), with same parametersapplied to all ADRP images. Pearson’s correlation coefficients andManders’ overlap coefficients were calculated for each scatterplot(Zinchuk and Zinchuk, 2008).

Electron microscopyVector-3 or D52-2-7 cells were fixed in 2% buffered glutaraldehyde(ProSciTech) for 2 h, washed in 0.1 M MOPS buffer, scraped from theculture flask, encapsulated in 10% BSA (Sigma-Aldrich), then cross-linkedwith glutaraldehyde to form a pellet. Small blocks were post-fixed in 2%buffered osmium tetroxide for 3 h followed by dehydration in a gradedethanol series. After infiltration in epoxy resin with acetone, cells wereembedded in epoxy resin (TAAB TLV) and polymerised for 10 h at 60°C.Semi-thin (500 nm) sections were cut on glass knives using a Leica UltracutUC6 ultramicrotome (Leica Microsystems, Vienna) and stained withMethylene Blue for light microscopic evaluation. Selected areas weretrimmed and ultrathin (70 nm) sections were cut with a diamond knife(Diatome, Switzerland), and collected onto 300 mesh thin bar coppergrids (ProSciTech). Grids were stainedwith 2%uranyl acetate in 50% ethanol,followed by Reynold’s lead citrate, and examined using a Philips CM120BioTWIN transmission electron microscope (FEI, The Netherlands) at100 kV. Images were collected using an SIS Morada digital camera.

Cellular lipid droplet quantificationAll image data intended for quantitative comparison were acquired using thesame sub-saturating settings on a single focal plane. Numbers of lipiddroplets per cell and diameters of each lipid droplet were quantified usingImage-Pro Plus Version 5.1 software (MediaCybernetics, Rockville, MD).Lipid droplet areas were calculated from measured diameters. Based onexpected lipid droplet sizes in non-adipocytes ranging from 0.2–1 μm(Suzuki et al., 2011), objects with diameters smaller than 0.3 μm werefiltered to reduce noise from potential non-specific staining. The intensityrange was set according to the BODIPY 493/503 fluorescence intensitydetected in one TPD52-expressing cell line, D52-2-7, and the sameparameters were then used to quantify lipid droplets in all 3T3 cell lines.Images of at least 10 panels per cell line were quantified in each of threeindependent experiments.

The dispersal of cellular lipid droplets was investigated using ADRP andDAPI images. Measurements were carried out using Metamorph softwareVersion 7.7 (Molecular Devices, Sunnyvale, CA). After thresholding andbackground subtraction, the locations of the centre of each cell nucleusand lipid droplet were used to calculate the distance between the centre of eachlipid droplet and that of the respective cell nucleus, based on Pythagoras’theorem.

Quantification of cellular TAG contentCellular total lipid was extracted using the Folch method (Folch et al., 1957)and TAG concentrations were measured using a GPO-PAP kit (Roche

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Diagnostics) according to the manufacturer’s instructions, and normalisedaccording to cellular protein levels.

Western blot analysesCells were lysed in 3% SDS lysis buffer as described previously (Boutroset al., 2003). Between 20 and 30 μg total protein extracts were resolvedby SDS-PAGE on 12.5% mini polyacrylamide gels or NuPAGE® Novex4–12% Bis-Tris mini gels (Life Technologies, VIC, Australia).

Affinity-purified rabbit polyclonal TPD52 (1:100) and TPD52L1 (1:100)antisera were raised in-house (Balleine et al., 2000; Boutros et al., 2003).Fatty acid synthase (FASN) mouse monoclonal antibody (A-5, 1:200) waspurchased from Santa Cruz Biotechnology (Dallas, TX). SCD1 (M38,1:1000) rabbit polyclonal and Syntaxin 6 (C34B2, 1:1000) rabbitmonoclonal antibodies were purchased from Cell Signaling Technology.ADRP (1:2000) guinea pig polyclonal antisera were obtained from ProgenBiotechnik. ARL1 (EPR10595, 1:2000) and ARFRP1 (EPR3899, 1:1000)rabbit monoclonal antibodies, mouse monoclonal anti-NSF (NSF-1, 1:2000)and rabbit polyclonal anti-SNAP23 (1:500) antibodies were purchased fromAbcam. Syntaxin 5 (1:1000) and VAMP4 (1:1000) rabbit polyclonalantibodies were purchased from Synaptic Systems (Goettingen, Germany).RAB5C rabbit polyclonal antibody (1:500) was purchased from Sigma-Aldrich, and has been previously shown by our laboratory to cross-react withother RAB5 isoforms (Shahheydari et al., 2014). Mouse monoclonal α-tubulin antibody (DM1A) (1:5000; Sigma-Aldrich) and mouse monoclonalglyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (6C5,1:10,000; Life Technologies; VIC, Australia) were used as loading controls.

Assessment of lipid metabolismCells were incubated for 4 h in GIBCO® DMEM (Life Technologies)containing 2% fatty-acid-free BSA, [3H]acetic acid (1 µC/ml; Perkin Elmer,VIC, AU), [1-14C]oleic acid (0.5 µC/ml; Perkin Elmer, VIC, Australia) and0.5 mM unlabelled oleate (Sigma-Aldrich) to determine exogenous fatty acidoxidation and esterification into TAG, and TAG synthesis from de novolipogenesis. Fatty acid oxidation was determined by measuring 14CO2 in theculture medium and acid soluble metabolite (ASM) production. To assess fattyacid incorporation from de novo lipogenesis or exogenous oleate into TAG,cellular lipids were extracted (Folch et al., 1957). After centrifugation ofchloroform-methanol extracts (v/v, 2:1) for 10 min at 1000 g, the lower phasewas collected and evaporated under a stream of nitrogen. Lipids were separatedusing thin-layer chromatography by loading the dissolved pellet and an internalTAG standard on silica plates (Merck Millipore, VIC, Australia), and thenimmersing plates in heptane, isopropyl ether and acetic acid (60:40:3). Plateswere subsequently sprayed with chlorofluorescein dye (0.02% w/v in ethanol)and lipids were visualised under long-wave UV light. TAG bandswere scrapedand radioactivity was counted. The rate of TAG synthesis from de novolipogenesis was calculated as 3H counts (dpm/min) in the TAG fraction per mgof protein of cell lysates, and the rate of TAG synthesis from exogenouslysupplied oleate was calculated as 14C counts (pmol/min) in the TAG fractionper mg protein of cell lysates. The fatty acid uptake rate was calculated as thesum of 14C counts (pmol/min) funnelled into oxidation (14CO2+ASM) and 14Ccounts (pmol/min) in the TAG fraction, per mg of protein of cell lysates.

Palmitic acid treatment and cell proliferation assaysParental, vector control or TPD52-expressing 3T3 cell lines were plated intriplicate in 96-well plates (3×103 cells/well). After 24 h, cells were treatedor not with 250 μM palmitic acid (Sigma-Aldrich) in fatty-acid-free BSA.MTT assays were performed at indicated time points as described previously(Roslan et al., 2014).

Plasmid constructsPlasmids encoding TPD52 bait protein, and TPD52, TPD52L1, RAB5Cand GOLGA5 prey proteins have been previously described (Byrne et al.,1998; Shahheydari et al., 2014). Bait constructs encoding human TIP47(Genbank NM_005817.4) and ADRP (Genbank NM_001122.3) weregenerated by subcloning respective EcoRI–XhoI fragments from pVP16-TIP47 and pVP16-ADRP, which were kind gifts from Paul D. Bieniasz (TheRockefeller University, New York, NY) (Eastman et al., 2009), into the

EcoRI and SalI sites of pAS2.1. Sanger sequencing confirmed that pAS2.1-TIP47 construct encodes full-length TIP47 (434 amino acids), whereaspAS2.1-ADRP encodes ADRP residues 1–415 (of 437 residues) due to aC>T mutation at nt 1456 (NM_001122.3) that produces a stop codon.

Yeast two-hybrid systemFor direct interaction testing, Saccharomyces cerevisiae strain Hf7c cells weretransformed with paired bait (pAS2.1) and prey (pACT2) constructs, andcultured at 30°C on solid synthetic drop-out (SD) medium lacking Leu and Trp(SD/−Leu−Trp) (Byrne et al., 1998). Bait–prey interactions were scoredaccording to the time (in days) until visible colony growth on solid SDmediumlacking His, Leu and Trp (SD/–His–Leu–Trp) (Boutros et al., 2003;Shahheydari et al., 2014). Bait or prey constructs paired with the opposingempty vector represented negative controls, whereas the interaction betweenTPD52 bait and TPD52L1 prey was used as a positive control.

Pulldown assaysPulldown assays employed glutathione S-transferase (GST) and His6-taggedrecombinant mouse Tpd52 proteins expressed from pGEX3X (Byrne et al.,1998; Sathasivam et al., 2001), and GST-tagged recombinant humanTPD52 expressed from pGEX6P-1 (Chen et al., 2013; Shahheydari et al.,2014). Purification of mouse or human TPD52 fusion proteins andrespective GST controls was performed as previously described (Chenet al., 2013; Shahheydari et al., 2014). Purified proteins were assessed bySDS-PAGE. Vector and TPD52-expressing 3T3 cells were lysed in lysisbuffer (150 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl pH 7.5, 0.1%deoxycholate, 1% Triton X-100) (Lopez-Verges et al., 2006). A total of150 μg of recombinant TPD52 and respective GST proteins were cross-linked to GSH–agarose (Glutathione Sepharose 4B, GE Healthcare) asdescribed previously (Shahheydari et al., 2014), and incubated with 3T3 celllysates. Matrices were washed extensively before bound proteins wereeluted into SDS sample buffer and subjected to western blot analyses.

Isolation of lipid droplets by cellular fractionationCellular lipid droplets were isolated essentially as previously described(Brasaemle and Wolins, 2006). Briefly, D52-2-7 cells were treated with400 µM oleic acid in fatty-acid-free BSA for 24 h and then lysed inhypotonic lysis medium [HLM; 40 mM Tris–HCl pH 7.4, 2 mM EDTA,20 mM sodium fluoride, and EDTA-free protease inhibitor cocktail tablets(Roche Applied Science, NSW, Australia)). After incubation on ice for10 min, cell lysates were briefly sonicated and subjected to centrifugation at1000 g for 10 min. The resulting supernatant with the floating fat layer wascollected as a non-nuclear fraction into an ultracentrifuge tube and gentlymixed with ice-cold HLM containing 60% sucrose (final 20% sucrose), thenoverlaid sequentially with 15% sucrose in HLM, 5% sucrose in HLM andHLM only to form discontinuous gradients. Gradients were centrifuged at28,000 g in a Beckman SW41Ti rotor for 45 min at 4°C, and the lipiddroplet fraction was collected from the top layer. Proteins from the lipiddroplet fraction were concentrated by precipitation with 20% trichloroaceticacid (TCA, Sigma-Aldrich) at −20°C overnight, and washed three timeswith 100% acetone. After air drying, the pellet was dissolved in lysis buffer[150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl pH 7.5, 1% Triton X-100,50 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mMphenylmethylsulfonyl fluoride (PMSF), and EDTA-free protease inhibitorcocktail tablets]. Protein concentrations were measured using a Pierce™BCA Protein Assay Kit (Thermo Scientific, VIC, Australia), and 15 μgprotein was subjected to western blot analyses.

Statistical analysisThe SPSS for Windows package (version 21; IBM) was used for most graphgeneration and statistical analyses. Values from lipid quantitation analyseswere expressed as means±s.e.m. of three independent experiments, andcomparisons between groups weremade using a two-tailed, unequal varianceStudent’s t-test. The Mann–Whitney U-test (GraphPad Prism 4, GraphPadSoftware, La Jolla, CA) was utilised to compare the dispersal of cellular lipiddroplets, and Pearson’s correlation and Manders’overlap coefficients werecalculated for quantitative analysis of immunofluorescence staining.

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AcknowledgementsWe thankDrEmmaKettle andDrRossBoadle (ElectronMicroscopyUnit,WestmeadHospital, Westmead, NSW, Australia) for electron microscopy assistance, andDr Enoch Tay (Westmead Millennium Institute, NSW, Australia) and AssociateProfessor Geraldine O’Neill (Kids Research Institute, NSW, Australia) for expertadvice and discussions. Professor Paul D. Bieniasz (The Rockefeller University,USA) kindly provided pVP16-TIP47 and pVP16-ADRP plasmids, and Dr EnricoTiacci (University of Perugia, IT) provided TPD52monoclonal antibody for this study.The Leica SP5 II in the CLEM Suite at the Kids Research Institute was supported bythe following grants: Cancer Institute New South Wales Research Equipment [grantnumber 10/REG/1-23], NHMRC [2009-02759], the Ian Potter Foundation [grantnumber 20100508], the Perpetual Foundation [grant number 730], RamaciottiFoundation [grant number 3037/2010], and the Sydney Medical School ResearchInfrastructure Major Equipment Scheme. Ultrastructural studies were performed inthe Electron Microscope Laboratory, Westmead, a joint facility of the Institute forClinical Pathology and Medical Research and the Westmead Research Hub.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsA.K., N.R. and Y.C. performed experiments, analysed data and wrote themanuscript. S.F., A.J.H. and D.W. performed experiments, and analysed data.L.C.C., A.D.-F., R.K.B., G.E.G. and B.K.S. provided advice regarding experimentaldesign and/or reagents. J.A.B. conceived of the study and wrote the manuscript. Allauthors read and approved the final manuscript.

FundingThis work was supported by a postgraduate scholarship from the Government ofMalaysia (to N.R.); DOOLEYS Bridging grant support (to A.K. and Y.C.); and bydonations to the Children’s Cancer Research Unit of the Children’s Hospital atWestmead.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.167692/-/DC1

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