Fig 2: ADAR1 is highly expressed in CRPCa. (A) Representative images and (B) quantification of immunohistochemical staining for ADAR1 in BPH and PCa tissues. (C and D) Western blot analysis of ADAR1 protein expression in different tissue types. (E) Reverse transcription-quantitative PCR and (F and G) western blot analysis of the mRNA and protein expression levels of ADAR1 in different cell lines. *P<0.05 and **P<0.01. ADAR1, double-stranded RNA-specific adenosine deaminase; BPH, benign prostatic hyperplasia; PCa, prostate cancer; CRPCa, castration-resistant PCa.

Fig 3: ADAR1-Knockdown inhibits the proliferation and induces the apoptosis of DU145 and PC3 cells. Western blot analysis was used to confirm ADAR1-knockdown in (A and B) DU145 and (C and D) PC3 cells after siRNA transfection. Proliferation of (E) DU145 and (F) PC3 cells was inhibited by ADAR1-knockdown. ADAR1-Knockdown promoted the apoptosis of (G and H) DU145 and (I and J) PC3 cells, which was analysed by flow cytometry using Annexin V-FITC/PI staining. Statistical results are presented as the mean ± SD of three independent experiments. *P<0.05 and **P<0.01. ADAR1, double-stranded RNA-specific adenosine deaminase; OD, optical density; si (RNA), small interfering; NC, negative control.

Fig 4: CircCDK17 inhibited the proliferation and cell cycle progression of PASMCs. (A) Western blotting analysis showing that overexpression (OE) of circCDK17 attenuated hypoxia-stimulated PCNA expression, n = 6. (B–C) EdU staining (B) and CCK8 (C) analysis showing that OE-circCDK17 attenuated hypoxia-induced cell proliferation and viability, n = 6. (D) Flow cytometry showing the effect of OE-circCDK17 on cell cycle, n = 6. (E) RNA pull-down combined mass spectrometry showing circCDK17 binds with PCNA specific peptide. (F) Effect of circCDK17 overexpression on PCNA ubiquitination level, n = 3. (G) Western blotting analysis showing that silencing of circCDK17 attenuated the effect of ADAR1 gene knocking-down on protein expression of PCNA, Cyclin A and CDK1, n = 6. (H–I) CCK8 analysis (H) and EdU staining (I) showing that silencing of circCDK17 abolished the effect of ADAR1 deficiency on cell viability and proliferation in the presence of hypoxia, n = 6. NOR, normoxia; HYP, hypoxia; NC, negative control; SI, siRNA; OE, overexpression of circCDK17; Statistical analysis was performed with two-way ANOVA followed by Dunnett's test; All values are presented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig 5: ADAR1-containing EVs are released into supernatant. (A) ADAR1-HiBiT was released into the cell culture medium. Extracellular HiBiT was detected in the supernatant by HiBiT assay. (B) Number and size distribution of the EVs acquired from the cell culture media. Representative results are shown. (C) Representative electron microscopy images of EVs acquired are shown. Scale bar, 100 nm. (D) Isolated EVs from the culture medium of Huh7-ADAR1-HiBiT cells were subjected to HiBiT assay. Higher luciferase activity indicated that these EVs contained ADAR1-HiBiT. Data are presented the mean ± SD of four independent experiments. *P<0.05. ADAR, adenosine deaminase acting on RNA-1; EV, extracellular vesicle.