Additional file 5 of EIF4A3-mediated biogenesis of circSTX6 promotes bladder cancer metastasis and cisplatin resistance
Creators: name: Wei, Wenjie
name: Liu, Kan
name: Huang, Xing
name: Tian, Shuo
name: Wang, Hanfeng
name: Zhang, Chi
name: Ye, Jiali
name: Dong, Yuhao
name: An, Ziyan
name: Ma, Xin
name: Wang, Baojun
name: Huang, Yan
name: Zhang, Xu
Publisher: name: figshare
Year: 2024
Subjects: Biophysics
Biochemistry
Cell Biology
Molecular Biology
Pharmacology
Environmental Sciences not elsewhere classified
Cancer

description: Additional file 5: Fig S1.CircSTX6 promotes the metastasis in BCa. A The knockdown and efficiency of circSTX6 in UMUC3 cells was detected by qRT-PCR. B The overexpression efficiency of circSTX6 in UMUC3 cells was detected by qRT-PCR. C Representative and quantified results of the Transwell migration and invasion assays in UMUC3 cells transfected with scramble, sh-circ#1, sh-circ#2, vector or circSTX6. Scale bar, 100 μm. D Cell migratory capabilities were assessed by wound healing assays after knocking down or overexpressing circSTX6 in UMUC3 cells. Scale bar, 400 μm. E CCK-8 assays showed the viability of BCa cells stably transfected with vector or circSTX6. F Edu assays depicted the proliferation rate of BCa cells stably transfected with scramble, sh-circ#1 or sh-circ#2. Scale bar, 200 μm. The data are presented as the means ± S.D. of at least three independent experiments. *P &lt; 0.05. P values are calculated by Student’s t test in B and C and one-way ANOVA in A and C. Fig S2. MiR-515-3p inhibits BCa migration and invasion in vitro. A-B The influence on cell migration and invasion abilities of EJ (A) and UMUC3 (B) cells transfected with miR-NC, miR-515-3p, anti-miR-NC or anti miR-515-3p. Scale bar, 100 μm. C-D Cell migratory capabilities were assessed in BCa cells transfected with miR-NC, miR-515-3p, anti-miR-NC or anti miR-515-3p by wound healing assays. Scale bar, 400 μm. E Wound healing assays showed the migration abilities in EJ cells transfected with scramble or sh-circ#1, and those co-transfected with anti-miR-NC or anti miR-515-3p. Scale bar, 400 μm. The data are presented as the means ± S.D. of at least three independent experiments. *P &lt; 0.05. P values are calculated by Student’s t test in A-D and one-way ANOVA in E. Fig S3. CircSTX6 enhances the SUZ12 expression in BCa. A The mRNA and protein level of SUZ12 in UMUC3 cells stably transfected scramble, sh-circ#1, sh-circ#2, vector or circSTX6. B The expression level of SUZ12 in the TCGA BLCA dataset. C Western blot were performed to evaluate the expression of SUZ12 in EJ and UMUC3 cells which were transfected with scramble or sh-circ#1, and those co-transfected with mock or SUZ12. The data are presented as the means ± S.D. of at least three independent experiments. *P &lt; 0.05. P values are calculated by Student’s t test in A (right) and one-way ANOVA in A (left).Fig S4. Knockdown of SUZ12 inhibits BCa migration and invasion in vitro. A Transwell assays showed the migration and invasion of UMUC3 cells transfected with mock, SUZ12, scramble or sh-SUZ12#1. Scale bar, 100 μm. B-C Cell migratory capabilities were assessed in EJ and UMUC3 cells transfected with with mock, SUZ12, scramble or sh-SUZ12#1 by wound healing assays. Scale bar, 400 μm. The data are presented as the means ± S.D. of at least three independent experiments. *P &lt; 0.05. Pvalues are calculated by Student’s t test in A-C. Fig S5. PABPC1 could facilitate the expression level of SUZ12 in UMUC3 cells. A The efficiency of PABPC1 knockdown or overexpression in UMUC3 cells was verified by qRT-PCR (left) and western blot (right). B qRT-PCR assay showed the expression of circSTX6 in UMUC3 cells transfected with the indicated plasmids. C qRT-PCR and western blot assays showed the expression of SUZ12 in UMUC3 cells transfected with the indicated plasmids. The data are presented as the means ± S.D. of at least three independent experiments. *P &lt; 0.05. P values are calculated by Student’s t test in A and C and one-way ANOVA in A and C. Fig S6. Silencing of circSTX6/SUZ12 complex promotes the chemosensitivity of BCa cells to CDDP. A Determination of IC50 values for CDDP treatment 24 h in EJ cells which were stably transfected with mock or SUZ12 vector. B Flow cytometry apoptosis assays were used to assess apoptosis rate of UMUC3 cells stably transfected with sh-SUZ12#1 after CDDP treatment. C Determination of IC50 values for CDDP treatment 24 h in EJ cells which were stably transfected with vector or circSTX6 plasmid. D Flow cytometry apoptosis assays were used to assess apoptosis rate of UMUC3 cells stably transfected with sh-circSTX6 after CDDP treatment. E Tumor sphere formation assays were used to assess the self-renewal capacity of BCa treated with CDDP. The scale bar indicates 100 μm. F-G Immunofluorescence staining showed the expression of γH2AX in BCa cells treated with CDDP. The scale bar indicates 20 μm. H-I Immunofluorescence staining showed the expression of γH2AX in BCa cells treated with CDDP. The scale bar indicates 20 μm. J qRT-PCR assay showed the relative expression levels of circSTX6 in the EJ and EJ-CDDP cell lines. K Determination of IC50 values for CDDP treatment 24 h in EJ-CDDP cells which were stably transfected with scramble, sh-circ#1 or sh-circ#2 vector. L-M Flow cytometry assay revealed the CDDP effect in EJ-CDDP cells transfected with scramble, sh-circ#1 or sh-circ#2. N Flow cytometry assay revealed the CDDP effect in EJ-CDDP cells transfected with vector or circSTX6, and those co-transfected with scramble or sh-SUZ12#1. The data are presented as the means ± S.D. of at least three independent experiments. *P &lt; 0.05. P values are calculated by Student’s t test in B, D, G, I and J and one-way ANOVA in M and N.