Additional file 1 of CircPTPRA blocks the recognition of RNA N6-methyladenosine through interacting with IGF2BP1 to suppress bladder cancer progression

Additional file 1: Figure S1. A. and B. The efficiencies of stable models of IGF2BP1 overexpression or knockout in EJ and T24T cell lines. C. Sanger sequencing of genomic DNAs to validate IGF2BP1 knockout in EJ and T24T cells. The mutation patterns on the two alleles are highlighted in red. The red arrow represents the cleavage site. D. CCK-8 assay was performed to evaluate cell viability in BC cells with stable overexpression or knockout of IGF2BP1. E. and F. Cell cycle analysis and representative images and quantification of transwell assay indicating the proliferation and invasion of EJ cells with stable overexpression or knockout of IGF2BP1. The data are the means ± SEM of three independent experiments. **, P < 0.01. Figure S2. A. RIP and qRT-PCR assays using an antibody specific for IGF2BP1 showing the interaction between circRNAs (circPTPRA, circSMARCA5) and IGF2BP1 in EJ cells with stable overexpression or knockout of IGF2BP1. B. The knockdown efficiency of circPTPRA in T24T cells transfected with circPTPRA siRNA or corresponding negative control were evaluated by qRT-PCR. C. Overexpression or knockdown of circPTPRA did not affect the IGF2BP1 expression levels in T24T cells. D. Overexpression or knockout of IGF2BP1 did not affect the circPTPRA expression levels in T24T cells. **, P < 0.01. Figure S3. A. The expression level of circPTPRA in 64 paired of BC and corresponding adjacent tissues was determined by qRT-PCR. GAPDH was used as a loading control. B. The relative expression of circPTPRA was detected using qRT-PCR in SV-HUC-1, RT4, UMUC3, EJ, T24T and 5637 cells. GAPDH was used as endogenous control. C. The correlation between the transcript levels of IGF2BP1 and circPTPRA by qRT-PCR in BC tissues (n = 64). D. Kaplan-Meier curves indicating overall survival of 64 bladder cancer patients with ow or high expression of circPTPRA (the patients were divided into high- and low-expression groups using the median value as the cut-off point, n = 64, p = 0.0009, log-rank test). E. Representative image (left) and quantification (right) of migration and matrigel invasion assays showing the invasion of EJ cells stably transfected with empty vector and circPTPRA. F. Representative images (left) and quantification (right) of migration and matrigel invasion assays showing the invasion of EJ cells upon ectopic expression of IGF2BP1 combined with circPTPRA overexpression. G. Cell cycle distributions in EJ cells stably transfected as indicated were presented by flow cytometry (The results are mean ± SEM of three experiments). H. Immunohistochemical staining and quantification showing the Ki-67 and CD31 expression within subcutaneous xenograft tumors formed by T24T cells stably transfected as indicated (n = 5 for each group). Scale bars: 100 μm. Pearson’s correlation coefficient analysis in Figure S3C. **, P < 0.01. Figure S4. A. and B. The volcano plots in EJ and T24T cells upon circPTPRA overexpression. Figure S5. A. Representative images and cell count of migration and invasion assays for circPTPRA overexpression EJ cells transiently transfected with MYC or the control. B. Cell cycle assay of circPTPRA overexpression EJ cells transiently transfected with MYC or the control. C. Representative images and cell counts of migration and invasion assays for circPTPRA overexpression EJ cells transiently transfected with FSCN1 or the control. D. Cell cycle assay of circPTPRA overexpression EJ cells transiently transfected with FSCN1 or the control. Data are presented as the means ± SEM of three independent experiments. **, P < 0.01.