Fig 2: Identification of SMARCD1 mRNA as a target regulated by miR‐99a‐5p in the GEM‐R‐BC cell line. (A) Venn diagram and in silico analysis of mRNA sequences showed that 16 putative target candidate genes of miR‐99a‐5p are key molecules for gemcitabine‐resistant BC. (B) The mRNA expression level of SMARCD1 in the parental BC and GEM‐R BC strains was measured by qRT‐PCR and (C) the protein expression level was measured by western blot. The expression level of SMARCD1 was significantly higher in the GEM‐R BC strain. n = 3. *P < 0.0001, Mann–Whitney U tests. The error bars indicate SEM. imagej was used for protein levels. (D) mRNA expression of SMARCD1 in miR‐99a‐5p transfectants was measured by qRT‐PCR and (E) protein expression was measured by western blot. The expression of SMARCD1 was lower than that in mock or miRNA control transfectants. n = 3. *P < 0.0001, Bonferroni/Dun method. The error bars indicate SEM. imagej was used for protein levels. (F) Presumed miRNA target sites in WT or deleted regions. (G) Dual‐luciferase reporter assay using vectors encoding putative miRNA target sites in WT or deleted regions. The luminescence intensity was significantly reduced by cotransfection of miR‐99a‐5p and a vector with the 3′‐UTR of WT. n = 3. *P < 0.0001, Bonferroni/Dun method. The error bars indicate SEM. (H) SMARCD1 mRNA expression in the BLCA cohort of TCGA. SMARCD1 expression in BLCA samples (n = 404) was compared with that in normal samples (n = 19). P < 0.0001, Mann–Whitney U tests. These experiments were repeated at least three times. GEM‐R BC, gemcitabine‐resistant bladder cancer; BLCA, bladder urothelial carcinoma; miRNA, microRNA; WT, wild‐type.

Fig 3: Relationship between SMARCD1 and cellular senescence in the GEM‐R BC cell line. Senescence induction by si‐SMARCD1 transfection was confirmed by β‐galactosidase staining. Quantification of β‐galactosidase staining in (A) GEM‐R‐BOY cells and (B) GEM‐R‐T24 cells. n = 6. *P < 0.005, Bonferroni/Dun method. The error bars indicate SEM. (C) A typical micrograph of (A) and (B). Scale bar, 100 μm. (D) Western blot confirming the expression of p21waf1/cip1 in GEM‐R BC cells transfected with si‐SMARCD1. Quantification of β‐galactosidase staining of (E) GEM‐R‐BOY cells and (F) GEM‐R‐T24 cells in miR‐99a‐5p transfection. n = 6. *P < 0.0001, Bonferroni/Dun method. The error bars indicate SEM. (G) A typical micrograph of (E) and (F). Scale bar, 100 μm. (H) Western blot confirming the expression of p21waf1/cip1 in GEM‐R BC cells transfected with miR‐99a‐5p. imagej was used for protein levels and quantification of β‐galactosidase staining. These experiments were repeated at least three times. GEM‐R BC, gemcitabine‐resistant bladder cancer; SA‐β‐gal, senescence‐associated beta‐galactosidase.

Fig 4: SMARCB1 is essential in maintaining the SWI/SNF complex integritya–b. Immunoprecipitation (IP) of the SWI/SNF complex subunits SMARCC1, SMARCA4, or ARID1A from the nuclear extracts of G401 (a) and BT16 (b) cell lines with or without Doxycycline (Dox)-induced SMARCB1 re-expression. Immunoblotted for subunits SMARCB1, SMARCC1, SMARCA4, ARID1A, ARID1B, SMARCC2, SMARCD1, SMARCE1, ACTL6A, and DPF2. Actin is a loading control.c. Mass spectrometry showing increased recovered peptides of SWI/SNF complex subunits by IP of SMARCA4 and SMARCC1 in G401 cells after SMARCB1 re-expression (Dox vs. No Dox).d. Glycerol sedimentation (10–30%) assay of SWI/SNF complex (~2MDa) from SMARCB1-deficient G401 cells without (top half) or with Dox (bottom half) immunoblotted for the indicated SWI/SNF complex subunits. BMI1 is a PRC1 complex subunit serving as a control.e. Immunoprecipitation (IP) of the SWI/SNF complex by SMARCC1 or SMARCA4 from the nuclear extracts of WT or Smarcb1 deficient mouse embryonic fibroblasts immunoblotted for the indicated SWI/SNF complex subunits.

Fig 5: Effect of SMARCD1 knockdown in GEM‐R BC cells. The knockdown efficiency of si‐SMARCD1 was verified by evaluating (A) the expression level of SMARCD1 mRNA measured by RT‐qPCR and (B) the SMARCD1 protein level measured by western blot analysis. n = 3. *P < 0.0001. The error bars indicate SEM. imagej was used for protein levels. (C) Cell proliferation by XTT assay. n = 6. *P < 0.01; **P < 0.0001; ns, not significant. The error bars indicate SEM. (D) Cell migration activity measured by wound healing assay. n = 4. *P < 0.01; **P < 0.0001; ns, not significant. The error bars indicate SEM. (E) Cell invasion activity measured by Matrigel invasion assay. Invasion cells were counted and compared. n = 6. *P < 0.05; **P < 0.01; ***P < 0.0001; ns, not significant. The error bars indicate SEM. All experiments were performed in quadruplicate and si‐SMARCD1 transfectants were compared with mock or si‐control transfectants. Transfection of si‐SMARCD1 increased the sensitivity of the GEM‐R BC cell line to gemcitabine. IC50 concentrations of gemcitabine were given to BC cells. Ten nanomolar si‐SMARCD1 transfection increased gemcitabine sensitivity in (F) GEM‐R‐BOY cells and (G) GEM‐R‐T24 cells, as determined by XTT assay. n = 6. *P < 0.0001. The error bars indicate SEM. The combination of si‐SMARCD1 transfection and gemcitabine administration had a clear additive effect on (H) GEM‐R‐BOY cells and (I) GEM‐R‐T24 cells and significantly inhibited cell proliferation. n = 6. *P < 0.001; **P < 0.0001. The error bars indicate SEM. The relationships between two groups were analyzed using Mann–Whitney U tests. The relationships between three or more groups were analyzed using the multiple comparison test with the Bonferroni/Dun method. These experiments were repeated at least three times. GEM, gemcitabine; GEM‐R BC, gemcitabine‐resistant bladder cancer.