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SUPPLEMENTARY FIGURE LEGENDS

Supplementary Figure 1. Detection of G-quadruplex by CD Spectra and native PAGE. (A)

Sequence of NRQ with G-tracts underlined (Top). CD spectrum of NRQ in the presence of

different concentrations of K+ (Left) or different monovalent cations (Right). (B) Sequence of

CRQ with G-tracts underlined (Top). CD spectrum of CRQ in the presence of different

concentrations of K+ (Left) or different monovalent cations (Right). (C) Sequences of I-8 and I-8

G4m with GG runs underlined and substitution mutations highlighted in blue (Top). CD

spectrum of I-8 G4m in the presence of different concentrations of K+ (Left) or different

monovalent cations (Right). (D) Native gel of 28 nucleotide I-8 and its mutant I-8 G4m, folded

in the presence of 100 mM K+ with or without TMPyP4. TMPyP4 unfolds I-8, allowing it to

migrate more quickly through the gel at a linear size.

Supplementary Figure 2. Effect of TMPyP4 on alternative splicing. (A) Fluorescent images

after transfection of HEK 293FT cells with I-8 or I-8 G4m constructs with or without presence of

TMPyP4 displaying inclusion (green), skipping (red), and merged (yellow). TMPyP4 promotes

exon skipping only in the I-8 transfection. (B,C) Semi-quantitative PCR (B) and qRT-PCR (C) of

transfections in Fig S2A showing significant increase in exon skipping in the presence of

TMPyP4. (D) Quantification of CD44v8-9/CD44s splicing ratio after titration of TMPyP4 with

or without cycloheximide. TMPyP4 promotes exon skipping regardless of cycloheximide,

suggesting that change in splicing ratio is not affected by nonsense-mediated decay (NMD).

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Supplementary Figure 3. G-quadruplexes stimulate variable exon inclusion. (A) Sequences

(top) and proposed G-quadruplex secondary structures (bottom) of G2U1, G3U1, NRQ and CRQ.

(B) CD spectra of G2U1, NRQ, G3U1, and CRQ oligonucleotides with 100 mM KCl. (C) CRQ

and CRQ mutant sequences (Left). Fluorescent images after transfection of HEK 293FT cells

with CRQ and mutant constructs showing mutants promote less exon inclusion (Right). (D, E)

Semi-quantitative RT-PCR (D) and qRT-PCR (E) of transfections in S3C. The CRQ G-

quadruplex but not the mutant sequences stimulate exon inclusion. (F) G2U1 and G2U1 G4m

mutant sequences (Top). Fluorescent images after transfection of HEK 293FT cells with G2U1

and mutant constructs showing mutant does not promote exon inclusion (Bottom left). Semi-

quantitative RT-PCR quantification of splicing of transfections in (Bottom right).

Supplementary Figure 4. hnRNPF is a potential G-quadruplex binding protein. (A)

Pairwise correlation and hierarchical clustering of hnRNP CLIP-seq datasets from Huelga et al.,

2012. hnRNPH shows weakest correlation with other hnRNPs. (B) Examples are shown for

hnRNPF CLIP-seq binding sites that contain predicted G-quadruplexes from mining hnRNPF

CLIP data and hnRNPF-regulated alternative exons identified in Huelga et al., 2012. Constitutive

exons are colored gray and the cassette exons are colored blue. hnRNPF binding regions are

depicted in yellow. Zoomed sequences display the full hnRNPF CLIP binding sequence with the

G-quadruplex guanines underlined and colored red.

Supplementary Figure 5. The functions of hnRNPF, ESRP1 and hnRNPH in splicing

regulation. (A) RNA pull down of biotinylated I-8 RNA probe incubated with recombinant

hnRNPF with increasing molar ratios of unlabeled I-8 or I-8 G4m oligos to biotinylated I-8

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probe, showing that only the I-8 oligos compete away hnRNPF binding. (B) Fluorescent images

after transfection of HEK 293FT cells with I-8 minigene constructs and increasing

concentrations of hnRNPF showing hnRNPF promotes exon inclusion. (C) Semi-quantitative

PCR of hnRNPF titration with I-8 and I-8 G4m showing dose-dependent increase in exon

inclusion by hnRNPF only with I-8 sequences (Right). (D) qRT-PCR after transient transfection

of I-8 minigene in HEK 293FT shNS or sh-hnRNPF cells results shows increased exon skipping.

*** = p<0.001, Student’s t-test. (E) Semi-quantitative PCR after transient transfection of I-8

minigene and hnRNPF with addition of increasing concentrations of TMPyP4. TMPyP4

promotes exon skipping in a dose dependent manner. (F) qRT-PCR showing effects of hnRNPF

and ESRPI on the alternative splicing of CD44 v8 minigene. hnRNPF and ESPR1 were

transiently transfected alone or in combination into HEK 293FT cells. (G) Overexpression of

ESRP1 in both control (shNS) and hnRNPF knockdown (shF) HEK 293FT cells promoted exon

inclusion of CD44 v8 minigene. (H) Overexpression of hnRNPF in both control (shNS) and

ESRP1 knocked down (shESRP1) HEK 293FT cells promoted exon inclusion of CD44 v8

minigene. (I) hnRNPF promotes stronger exon inclusion of CD44 v8 minigene compared to

hnRNPH after co-transfection into HEK 293FT cells.

Supplementary Figure 6. Predicted G-quadruplex enrichment near hnRNPF-regulated

cassette exons and semi-quantitative RT-PCR validation of splicing events. (A) Diagram of

cassette exon alternative splicing events and regions scanned for G3N7 predicted G-

quadruplexes (PGQs) within exons and 150 nucleotides proximal to splice sites flanking the

cassette exon (Top panel). Percentage of hnRNPF-regulated cassette exons containing PGQs

upstream, proximal, or downstream of the cassette exon compared to the same regions in non-

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hnRNPF-regulated cassette exons. p-value calculated by Fisher’s exact test (Bottom panel). (B-

D) Semi-quantitative RT-PCR images of hnRNPF-regulated cassette exons containing predicted

G-quadruplex structures located within 250 nucleotides downstream of cassette exon 5’ splice

site (B), 250 nucleotides upstream of cassette exon 3’ splice site (C), and within 250 nucleotides

upstream and downstream of the proximal cassette exons splice sites (D).

Supplementary Figure 7. hnRNPF regulates the expression of endogenous CD44 and EMT.

(A) Western blot images showing that silencing hnRNPF by shF2 promotes CD44 isoform

switching to CD44s (left panel), decreases the expression of epithelial markers E-cadherin and γ-

catenin, and increases the expression of mesenchymal markers N-cadherin and vimentin (right

panel) in MCF10A cells during TGF-β-induced EMT. (B) Morphology changes of MCF10A

control (shNS) and hnRNPF knockdown (shF) cells during TGF-β-induced EMT. (C) Western

blot images showing CD44 isoform switching to CD44s (left panel) and accelerated switching of

expression of EMT markers (right panel) by silencing hnRNPF (shF) in HMLE Twist-ER cells

during tamoxifen (4-OHT) induced EMT. (D) Immunofluorescence (left panel) and phase

images (right panel) showing the acceleration of loss of E-cadherin and gain of mesenchymal

morphology in hnRNPF silenced (shF) HMLE-twist ER cells during 4-OHT-induced EMT. (E)

Western blot analysis of CD44 splice isoforms and EMT markers in control and shF2-expressing

HMLE-Twist ER cells during 4-OHT-induced EMT. (F) Immunofluorescence (left panel) and

phase images (right panel) in control and shF2-expressing HMLE-Twist-ER cells during 4-OHT-

induced EMT. (G) Wound healing assays showing that silencing hnRNPF (shF) in HMLE cells

promoted cell migration. The representative images are shown on the left panel, and the line plot

of normalized scratched area percentage is on the right. Error bars represent S.E.M. ** = p <

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0.01, Student’s t-test. (H) Proliferation assay showing that hnRNPF overexpression did not affect

cell proliferation in Mes10A cells. Error bars represent S.E.M. N.S., non-significant by p > 0.05;

Student’s t-test.