Mazurek et al. Supplementary information

SUPPLEMENTARY EXPERIMENTAL PROCEDURES

Cell Culture and reagents:

The LS-LiM6 cell line, a derivative with high liver-metastasizing ability, was established from the LS174T colonic adenocarcinoma cell line by serially selecting cells that metastasized from cecum to liver as previously described [30]. The pair of human colon cancer cell lines DLD-1 and DLD1-TR was a kind gift from Dr. Bingliang Fang (MD Anderson Cancer Center) and the human cancer cell line GI-100 was a kind gift from Dr. Janet Price (MD Anderson Cancer Center). The selection of DLD1-TR TRAIL-resistant cells was previously reported [15]. The establishment of GI101-TR cell line was carried out following the same protocol as for LiM6-TR.

Cell lines were validated by STR DNA fingerprinting using the AmpF_STR Identifiler kit according to manufacturer's instructions (Applied Biosystems cat 4322288). The STR profiles were compared to known ATCC fingerprints (ATCC.org), and to the Cell Line Integrated Molecular Authentication database (CLIMA) version 0.1.200808 (http://bioinformatics.istge.it/clima/). The STR profiles matched known DNA fingerprints of LS174T colon cancer cells. Cells were maintained at 37oC in a 5% CO2 atmosphere in Dulbecco’s modified Eagle medium containing 10% heat-inactivated fetal bovine serum with penicillin and streptomycin.

Antibodies and inhibitors:

Anti-human galectin-3 was purified from culture medium of TIB166 rat hybridoma cells, obtained from the American Type Culture Collection. Antibodies purchased from Cell Signaling included anti-PARP (#9542), anti-Akt (#9272), anti-P-Akt (#4056), anti-PTEN (#9559), anti-phospho-PTEN (#9554), anti-ERK (#9102), anti-P-ERK (#9101), anti-JNK (#9165), anti-P-JNK (#9261), anti-beta-catenin (#9587), anti-caspase-8 (#9746), anti-caspase-9 (#9502), anti-caspase-3 (#9665), anti-Bid (#2002), anti-FADD (#2782), and anti-FAF1 (#4932). Goat anti-human DR4 (#AF347), goat anti-human DR5 (#AF631), and rabbit anti-human FLIP (#AF821) were from R&D Systems. HRP-conjugated anti-rabbit, anti-mouse, anti-goat and anti-rat secondary antibodies were from Millipore. Alexa Fluor 488 donkey anti-mouse IgG (#A-21202), Alexa Fluor 594 donkey anti-rat IgG (#A-21209), and Alexa Fluor 647 donkey anti-goat IgG (#A-21447) were from Invitrogen. The JNK inhibitor SP600125 and caspase-3 inhibitor Ac-DEVD-FMK were from Calbiochem.

Subcellular fractionation:

The release of cytochrome c into the cytoplasm of untreated and TRAIL-treated cells was determined in floating and attached cells combined using the APO Alert Cell Fractionation kit (Clontech). The quality of the separation of the mitochondrial and cytosolic fractions was determined by anti-COX4 antibody, and the location of cytochrome c was detected by Western blot using an anti-cytochrome c antibody.

Western blot:

Total cell lysate extraction and Western blots were performed as previously described [17]. Equal amounts of protein, measured using a Dc protein assay reagent (Bio-Rad), were separated by 12% SDS-PAGE then transferred electrophoretically to a nitrocellulose membrane. Immunoreactivity was detected by incubation with primary antibodies detailed above followed by horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence (ECL) reagents using the ECL detection system (Amersham Biosciences). Anti-beta-actin (Sigma #9587) was used to monitor equal loading in each lane.

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SUPPLEMENTARY DATA.

Figure S1. Restoration of TRAIL dependent loss of cell-surface DR4 in LiM6-TR cells treated with galectin-3 sh-RNA.

LiM6-TR cells were treated with galectin-3 sh-RNA (TR-SH-G) or non-targeting control sh-RNA (TR-C-SH) lentiviral particles. Cell surface DR4 death receptor expression, was determined by flow cytometry with (red) or without (blue) pretreatment treatment with 100 ug/ml TRAIL (30 min at 37oC). Background staining (no anti-DR4), grey).

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LS-LiM6 LiM6-TR TR-G-SH

Control

TRAIL

Figure S2. Restoration of TRAIL dependent internalization of cell-surface DR4 in LiM6-TR cells treated with galectin-3 sh-RNA.

LiM6-TR cells were treated with galectin-3 sh-RNA (TR-SH-G) or non-targeting control sh-RNA (TR-C-SH) lentiviral particles. Cells were pre-labeled with anti-DR4 and exposed to 100 ug/ml TRAIL on ice for 1 h. The temperature was raised to 37oC for 30 min to allow receptor internalization or kept at 0oC for controls. Surface labeling was removed by treatment with 2 mM acetic acid, then the cells were fixed and permeabilized. Internalization of death receptor immuno-complexes (red) was visualized with Alexa Fluor-conjugated secondary antibody and nuclear staining by DAPI (blue) by confocal microscopy. Only data for DR4 is shown; similar images were captured for DR5.

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DLD-1GI101-TRDLD1-TR

GI-1010

10

20

30

40

50%

sub

-G1

untreatedTRAIL

A

Surface Galectin-3

Cel

l Num

ber DLD1-TR

DLD-1C

Surface Galectin-3

Cel

l Num

ber

GI-101

GI101-TR

D

Galectin-3

Beta-actin

B

DLD-1GI101-TRDLD1-TR

GI-101

Figure S3. Surface expression of galectin-3 is elevated in DLD1-TR and GI101-TR cells.

A. TRAIL-dependent apoptosis of parental DLD1 colon cancer cells and GI-101 breast cancer cells was determined by flow cytometry of PI-stained cells after exposing them to 100 ng/ml TRAIL for 24 hrs. TRAIL resistance of DLD1-TR and GI101-TR was confirmed in the same analysis. B. Western blot analysis of galectin-3 expression in total cell lysates of DLD-1, DLD1-TR, GI-101, and GI101-TR. C. Cell surface expression of galectin-3 in DLD-1 (blue) and DLD1-TR (red) was monitored by FACS analysis. Control staining, grey. D. Cell surface expression of galectin-3 in GI-101 (blue) and GI101-TR (red) was monitored by FACS analysis. Background staining, grey.

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