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Supplementary Figure S1 FPKM ESRP1ESRP2 normaltumor Supplementary Figure S1. ESRP1 and ESRP2 mRNA expression in 65 normal kidney samples and 474 ccRCC.

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Presentation on theme: "Supplementary Figure S1 FPKM ESRP1ESRP2 normaltumor Supplementary Figure S1. ESRP1 and ESRP2 mRNA expression in 65 normal kidney samples and 474 ccRCC."— Presentation transcript:

1 Supplementary Figure S1 FPKM ESRP1ESRP2 normaltumor Supplementary Figure S1. ESRP1 and ESRP2 mRNA expression in 65 normal kidney samples and 474 ccRCC tumor samples using TCGA ccRCC RNA-seq data. FPKM: fragments per kilobase of exon per million mapped sequence reads.

2 a TSC2 Relative expression Normalized to GAPDH siNC siArkadia SLK Relative expression Normalized to GAPDH siNC siArkadia * ITGA6 Relative expression Normalized to GAPDH siNC siArkadia * ENAH Relative expression Normalized to GAPDH siNC siArkadia b Supplementary Figure S2 Supplementary Figure S2. qRT-PCR analyses of ITGA6, SLK, and TSC2 mRNA expression normalized to GAPDH in HEK293T cells (a), and ENAH mRNA expression normalized to GAPDH in OS-RC-2 cells (b). *P < 0.05 by two-sided Student’s paired t-test. siNC: negative control siRNA. n = 3. HEK293T cells OS-RC-2 cells

3 Supplementary Figure S3 a b Relative expression hrs ESRP2ArkadiaSmad7 hrs Relative expression ESRP2ArkadiaSmad7 Supplementary Figure S3. qRT-PCR analyses of ESRP2, Arkadia, and Smad7 expression after TGF-  stimulation (1 ng/ml) in HEK293Tcells (a) and OS-RC-2 cells (b). Experiments were repeated, and a representative set of data is shown.

4 Anti-HA Anti-Myc ESRP2-HAWT RRM2-KR RRM3-KR 6Myc-Arkadia-WT + + + + + + + + + + + + + + + CHX (h) 0 1 3 6 10 0 1 3 6 0 1 3 6 Supplementary Figure S4 Supplementary Figure S4. Cycloheximide chase assay. HEK293T cells over-expressing Arkadia and ESRP2 wild-type (WT) and KR mutants were harvested temporally after adding cycloheximide (50 μg/ml), followed by SDS-PAGE and immunoblotting.

5 Relative expression Normalized to GAPDH Relative expression Normalized to GAPDH ESRP2Arkadia siNC siArkadia#1 siESRP2#3 siNC siArkadia#1 siESRP2#3 Supplementary Figure S5 a Arkadia Relative expression Normalized to GAPDH siNC siArkadia#1 siESRP2#2 Relative expression Normalized to GAPDH ESRP2 siNC siArkadia#1 siESRP2#2 b Supplementary Figure S5. qRT-PCR analyses of ESRP2 and Arkadia mRNA expression in OS- RC-2 cells (a) and HEK293T cells (b) under the knockdown of ESRP2 or Arkadia used for qRT- PCR (Figures 5c and d). Values were normalized to GAPDH. siNC: negative control siRNA. Experiments were repeated, and a representative set of data is shown.

6 a siESRP2#1 +siArkadia#1 siESRP2#2 +siArkadia#2 siESRP2#2 siArkadia#1 siArkadia#2 siNC Relative expression Normalized to GAPDH siESRP2#1 +siArkadia#1 siESRP2#2 +siArkadia#2 siESRP2#2 siArkadia#1 siArkadia#2 siNC Relative expression Normalized to GAPDH b de Anti-Arkadia Anti-ESRP2 Anti-  -tubulin siESRP2#1 +siArkadia#1 siESRP2#2 +siArkadia#2 siESRP2#2 siArkadia#1 siArkadia#2 siNC ESRP2 c Relative expression Normalized to GAPDH siNC siESRP2#2 siArkadia#1 siESRP2#2 +siArkaadia#1 Relative expression Normalized to GAPDH siNC siESRP2#2 siArkadia#1 siESRP2#2 +siArkaadia#1 Supplementary Figure S6 Arkadia ESRP2 ArkadiaESRP2 Supplementary Figure S6. Expression of Arkadia and ESRP2 upon knockdown by siRNAs used for cell counting (Figures 5e and f). (a and b) Expression of Arkadia (a) and ESRP2 (b) mRNA in HEK293T cells under the knockdown of ESRP2 and/or Arkadia. (c) Arkadia and ESRP2 protein expression in HEK293T cells under the knockdown of ESRP2 and/or Arkadia. (d and e) Expression of Arkadia (d) and ESRP2 (e) mRNA in OS-RC-2 cells after cell counting. siNC: negative control siRNA. Experiments were repeated, and a representative set of data is shown.

7 Supplementary Figure S7 NormalTumor ab Anti-Arkadia Anti-  -tubulin Anti-N-cadherin Anti-E-cadherin Anti-  -tubulin siNC siArkadia 175 58 175 58 Supplementary Figure S7. Expression of E-cadherin and N-cadherin in ccRCC. (a) Log2 (E-cadherin expression/N-cadherin expression) in 65 normal kidney samples and 474 ccRCC tumor samples using TCGA ccRCC RNA-seq data. (b) Immunoblot analyses of E-cadherin and N-cadherin protein expression with negative control siRNA or siArkadia in OS-RC-2 cells.  -tubulin was used as a loading control. siNC: negative control siRNA.

8 Migrated cells (cell number per field) siNC siArkadia Supplementary Figure S8 Supplementary Figure S8. Transwell cell migration assay in OS-RC-2 cells treated with siRNA for Arkadia. siNC: negative control siRNA. Experiments were repeated, and a representative set of data is shown.

9 Supplementary Figure S9 0 5 10 15 ESRP2 expression (FPKM) Arkadia low Arkadia high Supplementary Figure S9. Dot blot of ESRP2 mRNA expression using TCGA ccRCC RNA-seq data. Patients were equally divided into two groups based on the expression of Arkadia mRNA. n = 466.

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