Fig. 4

Transcriptional activation of endogenous CDK13 upregulates E2F5 expression by promoting circCDK13 formation. a, PC3 cells were transfected with CDK13 sgRNA, oeCDK13 or their respective control vector, and then Western blot analysis detected the CDK13 and E2F5 expression. b, PC3 cells were treated as in (A), RT-qPCR detected E2F5 mRNA expression. c and d, PC3 cells were transfected with shCDK13 or pLKO or treated with CDK13 inhibitor THZ531, and then Western blot (C) and RT-qPCR (D) analysis detected the CDK13 and E2F5 expression. e and f, PC3 cells were transfected with CDK13 sgRNA or NC sgRNA and treated with actinomycin D (Act D). Western blotting (E) detected E2F5 protein level, and RT-qPCR (F) detected the expression of circRNAs (hsa_circ_0001699, hsa_circ_0079929, hsa_circ_0079933 and hsa_circ_0079939). *P < 0.05 vs. NC sgRNA+DMSO; #p<0.05 vs. CDK13 sgRNA+DMSO. g, PCR was used to detect hsa_circ_0079929 (termed circCDK13) in PCa tissues by using convergent or divergent primers. Divergent primers amplify circCDK13 in cDNA but not in genomic DNA (gDNA). GAPDH was used as linear control. h, RT-PCR amplified full-length circCDK13 in PC3 cells, and the amplified products were confirmed by agarose gel electrophoresis. i, Sanger sequencing confirmed head-to-tail splicing of circCDK13. j, RT-qPCR detected the expression of circCDK13 in PCa and BPH tissues. **P < 0.01 vs. BPH. k, The correlation between circCDK13 and E2F5 mRNA expression in our clinical data was analyzed by Pearson correlation analysis (R = 0.3976, P = 0.0296). l, PC3 and 22RV1 cells were transfected with si-circCDK13, si-linear CDK13 or si-Ctl. RT-qPCR detected circCDK13 and CDK13 mRNA expression. Bars are mean ± SEM of triplicate samples. *P < 0.05, **P < 0.01 vs. si-Ctl. m, RT-qPCR examined the expression of CDK13 mRNA and circCDK13 in PC3 and 22RV1 cells transfected with circCDK13 or empty vector. *P < 0.05, **P < 0.01 vs. empty vector. n, Western blotting detected the E2F5 expression in PC3 and 22RV1 cells transfected with si-circCDK13, circCDK13 or their respective control. o, PC3 and 22RV1 cells were transfected with circCDK13 and shE2F5 either alone or together, and then colony formation assay was performed to detect the cell proliferation. *P < 0.05 vs. pLKO+pWPI, #p<0.05 vs. circCDK13 + pLKO