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Additional file 1. Figure S1. Smyca expression correlates with poor prognosis. (A-F) Kaplan-Meier analysis of Smyca expression in relation to the overall survival (A, C, E, F) and disease-free survival (B, D) of indicated cancer types. Data were retrieved from TCGA data sets (A, B) or GEO data sets (C, D, E), or generated from an in house cohort (F). Patients were grouped into high and low expression based on the median expression level. Patients without survival information were omitted. Hazard ratio (HR) and P values are determined by log-rank test. (G) Smyca expression in HCC tumor tissues and adjacent normal tissues analyzed by qRT-PCR. (H–K) The correlation of Smyca expression with the stage (H, J) and invasiveness (I, K) of indicated cancer types. Data in (G-K) are presented by Whiskers boxplot, whiskers: min to max, bound of box: lower and upper quartiles, center line: median. Data points derived from basal-like subgroup of patients are marked in blue in (K). P values in (G), (H), (I), (J), (K) are determined by unpaired t-test. (L) Smyca expression in different subtypes of breast cancer patients analyzed by qRT-PCR. Data are expressed as mean ± SD. P values are determined by oneway ANOVA with Tukey’s post hoc test. Figure S2. Smyca promotes EMT. (A) Smyca expression in different breast cancer and HCC cell lines analyzed by qRT-PCR and represented as copy numbers. Data are expressed as mean ± SD from three independent experiments. (B) qRT-PCR analysis of indicated miRNAs in MDA-MB-231 cells stably expressing various Smyca shRNAs. Data are normalized with that of control cells and are expressed as mean ± SD from three independent experiments, ns, not significant by one-way ANOVA with Tukey’s post hoc test. (C, D) Western blot analysis of EMT markers in Hs578T cells stably expressing Smyca shRNAs (C) or MCF7 cells stably overexpressing Smyca (D). The amounts of each protein in relation to the control cells are indicated under the bands. Smyca expression levels in these stable lines are shown on the left panels. Data are mean ± SD from three independent experiments. (E-H) Cell proliferation (E, F) and cell viability (G, H) assays of M10 cells stably expressing Smyca (E, G) or MDA-MB-231 cells stably expressing Smyca shRNAs (F, H). Data are mean ± SD, n=3. P values are determined by unpaired t-test (E, G) or one-way ANOVA with Tukey’s post hoc test (F, H), ns, not significant. Figure S3. Bioinformatics and cell-based analysis for the relation of Smyca to TGF-β signaling. (A) Summary of the GSEA analysis for the match of Smyca signature with the indicated signatures. Data origin indicates the source database or the first author identifying the gene signature. (B) Intersection of Smyca-induced DEGs derived from MDA-MB-231 cells with Smad2/3 ChIP-seq data derived from Hs578T cells and BT-549 cells. The full list of overlapped genes is shown in Table S5. (C) qRT-PCR analysis for the expression of indicated TGF-β target genes in Hs578T cells stably expressing Smyca shRNAs and treated with or without 5 ng/ml TGF-β for 48 hr. Data are normalized with that of untreated group in each cell. (D, E) Luciferase assay for the Smad-target reporters transfected into Hs578T cells (D) or Malaru cells (E) stably expressing Smyca shRNAs and treated with or without 5 ng/ml TGF-β for 24 hr. The Smyca knockdown efficiencies are shown on the left panel in (E). Data in (C), (D), (D) are normalized with that of untreated control and presented as mean ± SD, n=3. P values are determined by one-way ANOVA with Tukey’s post hoc test, **P < 0.01, ***P < 0.001. (F, G) Summary of the correlations of Smyca expression with the expression of indicated TGF-β target genes by analyzing HCC or breast cancer data sets from TCGA (F) or GEO (G) databases. Pearson’s coefficients and P values are indicated. (H) GO analysis using the set of TGF-β target genes with expression levels showing positive correlations with Smyca expression in HCC and/or breast cancers. Figure S4. Smyca binds MH1 domains of Smad3 and Smad4 without affecting their expression and Smad3 phosphorylation. (A) Western blot analysis of Smad3 and Smad4 expression in MDA-MB-231 cells stably expressing Smyca shRNAs. (B) Western blot analysis for Smad3 phosphorylation in MDA-MB-231 cells stably expressing Smyca shRNAs and treated with or without 5 ng/ml TGF-β for 30 min. The blots are representative of three independent experiments and quantitative data are shown on the right. (C) Immunoprecipitation analysis of Smad3 and Smad4 interaction in MDA-MB-231 cells stably expressing indicated Smyca constructs and treated with or without 5 ng/ml TGF-β for 1 hr. (D) Luciferase assay for the Smad-responsive reporter in MDA-MB-231 cells transfected with indicated Smyca constructs and treated with or without 5 ng/ml TGF-β for 24 hr. The expression levels of Smyca are shown on the left panel. (E) Baculovirally purified Smad3, Smad4, and their MH1 deletion mutants bound on beads were incubated with biotinylated sense or antisense Smyca. The bound Smyca was analyzed by qRT-PCR. The equal inputs of recombinant proteins are shown on or antisense Smyca. The bound Smyca was analyzed by qRT-PCR. The equal inputs of recombinant proteins are shown on the right. Data in (B), (D) and (E) are mean ± SD, n=3. Pvalues are determined by one-way ANOVA with Tukey’s post hoc test (B, D) or unpaired t-test (E), *** P < 0.001; ns., not significant. Figure S5. Smyca is a Smad target and mediates a positive feedback control of TGF-β signaling. (A) qRT-PCR analysis of Smyca expression in MDA-MB-231 cells stably expressing Smad3 or Smad4 shRNAs and treated with or without 5 ng/ml TGF-β for 24 hr. The knockdown efficiencies of Smad3 and Smad4 shRNAs are shown on the left and middle panels, respectively. (B) qRT-PCR analysis of TGF-β target gene expression in MDA-MB-231 cells stably expressing Smyca shRNAs and treated with or without 5 ng/ml TGF-β for indicated time points. Data are normalized with that of untreated group (0 h). Data in (A) and (B) are mean ± SD, n=3. P values are determined by unpaired t-test (A) or one-way ANOVA with Tukey’s post hoc test (B), *** P < 0.001. Figure S6. Smyca promotes c-Myc transcription activity without affecting its expression or interaction with Max. (A) qRT-PCR analysis of the expression of indicated c-Myc target genes in Malaru cells stably expressing control or Smyca shRNA. Data are normalized with that of control cells. (B, C) Luciferase assay for a c-Myc-reponsive reporter transfected into MDA-MB-231 cells stably expressing Smyca or mutant (B) or Smyca shRNAs (C). The expression levels of Smyca and its mutant are shown on the left panel in (B). (D) Summary of the correlations of Smyca expression with the expression of indicated c-Myc target genes by analyzing breast cancer and HCC data sets from TCGA. Pearson’s coefficients and P values are indicated. (E) Maping the Smyca-binding region in c-Myc. Top: Schematic presentaton of c-Myc domains. The various GFP-c-Myc truncated proteins were purified from transfected 293T cells and incubated with biotinylated sense or antisense Smyca. The bound Smyca was analyzed by qRT-PCR. Data are normalized with that from GFP only control. The input levels of various GFP fusion proteins are shown on the right and marked by arrows. (F) Western blot analysis of c-Myc expression in indicated cells stably expressing Smyca or Smyca shRNAs. (G) Immunoprecipitation analysis of c-Myc/Max complex formation in MDA-MB-231 cells stably expressing Smyca or Smyca shRNA. Data in (A), (B), (C), (E) are mean ± SD, n=3. P values are determined by unpaired t-test (A), or one-way ANOVA with Tukey’s post hoc test (B, C, E), *P < 0.05, **P < 0.01, *** P < 0.001. Figure S7. Smyca-promoted c-Myc singaling neutralizes the growth inhibitory effect of Smyca-promoted TGF-β signaling. (A) Immunoprecipitation analysis of c-Myc/Smad complex formation in MDA-MB-231 cells stably expressing Smyca and treated with 5 ng/ml TGF-β for 2 hr. (B, C) Luciferase assay for a Smad-responsive reporter (B) or c-Myc-responsive reporter (C) transfected into MDA-MB-231 cells together with indicated Smyca constructs and treated with or without 5 ng/ml TGF-β for 24 hr. The expression levels of Smyca (1-500) and (1001-1500) fragments are shown on the left and middle panels in (B), respectively. (D, H) Cell proliferation assay of MCF7 cells stably expressing Smyca and treated with 75 μM 10058-F4 for 24 hr (D) or NTU-BL cells stably expressing Smyca and treated with 5 μM SB431542 or 75 μM 10058-F4 for 24 hr (H). (E, F) ChIP analysis for Smad3, Smad4, and c-Myc binding to the promoter regions of CDKN2B (E) and CDKN1A (F) genes in MDA-MB-231 cells stably expressing Smyca and treated with 5 μM SB431542 and/or 150 μM 10058-F4 for 2 hr. (G, I) qRTPCR analysis of the expression of indicated genes in MCF7 (G) or NTU-BL (I) cells stably expressing Smyca and treated with 5 μM SB431542 or 75 μM 10058-F4 for 24 hr. Smyca expression levels are shown on the left panel of (I). Data in (B), (C), (D), (E), (F), (G), (H), (I) are mean ± SD, n=3. P values are determined by one-way ANOVA with Tukey’s post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001. Figure S8. Downregulation of the expression of Smyca, Smad targets, and c-Myc targets by Smyca gapmer ASO. (A, B, D) qRT-PCR analysis of Smyca or indicated mRNAs in MDA-MB-231 cells transfected with Smyca gapmer ASO (A), LM6 cells treated with indicated doses of NPs carrying Smyca gapmer ASO or control gapmer (B), or LM6 tumor-bearing mice treated with NPs carrying Smyca gapmer ASO or control NPs (D). (C) The morphology, size and weight of primary tumors taken from the sacrifice day for experiment shown in Fig. 7F. Data in all panels are mean ± SD, n=3 (A, B, D) or 4 (C). P values are determined by unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Table S1. Antibody details. Table S2. siRNA, shRNA and Gapmer sequences. Table S3. Sequences of PCR primers. Table S4. ChIRP probe sequences. Table S5. List of genes that are regulated by Smyca and bound by Smad2/3. |
