Fig 2: PRPF6 promotes cell growth under androgen-depleted condition. A. Impacts of lentivirus-mediated PRPF6 knockdown on the protein expressions in LNCaP-AI cells. LNCaP-AI cells were infected with shPRPF6 or shCtrl, and then were collected for protein extraction and subjected to western blot analysis using antibodies using anti-PRPF6 (Proteintech), anti-AR (Proteintech) and GAPDH. B. PRPF6 knockdown repressed cell proliferation of LNCaP-AI cells. LNCaP-AI cells (2500 per well) with shPRPF6 or shCtrl were cultured in medium with 5% CSS. After indicated time, MTS reagent was added, then absorbance at 490 nm was measured and plotted. Data were means ± SD of three independent experiments. Student's t-test was performed. C. PRPF6 knockdown inhibited cell growth of LNCaP-AI cells under androgen-depleted condition. LNCaP-AI cells (5000 pre well) with shPRPF6 or shCtrl were infected with shPRPF6 or shCtrl, and then were cultured for 14 days. D. PRPF6 knockdown inhibited cell growth in xenograft mice under castration condition. Castration operation was performed on 5-week-old male NOD/SCID mice. After one week, CWR22Rv1 cells with shPRPF6 or shCtrl were injected subcutaneously into castrated male mice. Representative photograph of mice with tumor xenografts (upper panel). Photograph of all xenograft tumors derived from CWR22Rv1 cells with shPRPF6 or shCtrl (lower panel). E. Tumor volumes of xenografts in castrated male mice were shown. The volume of xenograft tumors derived from CWR22Rv1 cells with shPRPF6 or shCtrl were measured every 6 days. Data represent means ± SD. Student's t-test was performed. F. Final tumor weights of xenograft tumors derived from CWR22Rv1 cells with shPRPF6 or shCtrl in castrated male mice were measured and shown. Horizontal lines represent the mean values. Student's t-test was performed. G. Expressions of indicated proteins in xenograft tumors were detected by IHC assays. Scale bars, 100 µm.

Fig 3: Pre‐mRNA processing factor 6 (PRPF6) depletion inhibits hepatocellular carcinoma (HCC) cell growth and proliferation. A, B, Detection of knockdown efficiency of PRPF6 shRNA in HCCLM3 and PLC5‐androgen receptor (AR) cells. C, D, Knockdown of PRPF6 inhibits cell proliferation in HCC cells by MTS assay. *P < .05, **P < .01. E‐G, Knockdown of PRPF6 significantly inhibits cell growth in HCCLM3 and PLC5‐AR cells, but not in AR‐negative PLC5 cells by colony formation assay. H, PRPF6 knockdown delayed G1‐S phase transition in HCCLM3 cells, by flow cytometry assay. I, PRPF6 knockdown has no effect on cell migration in HCCLM3 cells, by scratch assay. J, PRPF6 depletion has no obvious effect on cell migration in PLC5‐AR cells, by Transwell assay. DHT, dihydrotestosterone; EtOH, ethyl alcohol; shCtrl, control

Fig 4: Pre‐mRNA processing factor 6 (PRPF6) promotes androgen receptor (AR) gene self‐transcription in hepatocellular carcinoma (HCC) cells. A, Quantitative PCR analysis was undertaken to detect mRNA expression of AR gene in HCC cells. ***P < .001. B, AR protein expression was induced by dihydrotestosterone (DHT) treatment in AR‐positive HCC cells. C, Ectopic expression of PRPF6 enhances AR protein level in a dose‐dependent manner. D, Schematic diagram of 2 androgen responsive elements (ARE) sites in the AR gene. E, Recruitment of AR or PRPF6 to ARE I/II regions on the AR gene. F, PRPF6 depletion reduced the recruitment of AR and histone H3K36me3 level at ARE I region of the AR gene. EtOH, ethyl alcohol; Ex, exon; siCtrl, control

Fig 5: Pre‐mRNA processing factor 6 (PRPF6) depletion inhibits hepatocellular carcinoma (HCC) tumor growth in mouse xenograft. A, Representative photographs showing xenograft tumors in male BALB/C‐null mice. Left, control (shCtrl); right, shPRPF6. B, PRPF6 depletion significantly inhibits tumor growth in vivo. C, PRPF6 depletion significantly reduces HCC tumor burden. ***P < .001. D, PRPF6 depletion significantly decreases HCC tumor weight. **P < .01. E, Expression of PRPF6 and Ki‐67 in xenograft tumors was analyzed by immunohistochemistry (200×). F, Protein expression of PRPF6 in xenograft tumors was detected by western blotting. G, Schematic representation of upregulation function of PRPF6 on AR self‐transcription, thereby coactivating androgen receptor (AR)/AR variant (AR‐Vs)‐induced transactivation to promote the progression of HCC. ARE, androgen responsive element; DHT, dihydrotestosterone