Fig 2: ROCK2 silencing reduces YAP expression and YAP-mediated transcriptional activity. a Immunofluorescence staining of YAP in U-2OS cells after 24 h of exposure to the Stemolecule™ ROCK2 Inhibitor (10 μM) or siRNA sequences targeting ROCK2 (siROCK2) or irrelevant target sequences (SCR). Digital images were taken under identical conditions using the image analysis software NIS-Elements (Nikon Italia); scale bar, 20 μm. b Western blotting of YAP in cytoplasmic and nuclear fractions of U-2OS cells after 24–48 h of exposure to ROCK2 inhibitor together with densitometric analysis. YAP signal was quantified against GAPDH or LAMIN B and reported as ratio of adjusted volume optical density (OD/mm2). Data are presented as the mean ± standard error (SE) of three separate experiments (** p < 0.01, Student’s t test) c qPCR analysis of the expression of CYR61, CTGF and CCND1 in U-2OS parental cells after 24-h to 72-h treatments. Data are shown as 2-ΔΔCt. GAPDH was used as a housekeeping gene. Data are presented as the mean ± standard error (SE) of three separate experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t test)

Fig 3: Decreased CDC42 induces entosis by promoting the ROCK1/2 signaling pathway and E-cadherin expression in Pca cells (×400 magnification). (A) No entosis morphology or increased ROCK1/2 or E-cadherin expression was observed in Pca cells treated with Akt inhibitor (MK2206; 10 µM) or ERK1/2 inhibitor (SCH772984; 3 µM) for 4 weeks, respectively. (B) IC50 values of the CDC42 inhibitor ML141 in the Pca cell lines. (C) Entosis-like morphology of Pca cells under ML141 pressure (2 µM for 4 weeks). (D) siRNA knockdown of CDC42 expression at the mRNA and protein level. *P<0.05 vs. control (Student's t-test) (E) CDC42 siRNA and ML141 (2 µM) promote ROCK1/2 expression and E-cadherin expression (following treatment for 1 week). *P<0.05 and **P<0.01 vs. control (one-way analysis of variance followed by Fisher's least-significant difference test). CDC42, cell division cycle 42; ROCK, Rho kinase; Pca, prostate cancer; Akt, protein kinase B; ERK, extracellular-signal-regulated kinase; IC50, half-maximal inhibitory concentration; siRNA, short interfering RNA; E-cadherin, epithelial cadherin; C, control; T, treatment; M, ML141 treatment; S/Si, siRNA treatment; Sc, scrambled.

Fig 4: Effects of NaHS on ROCK2 expression and activity as well as phosphorylation of ROCK2 at Tyr722 in RHNs. (A) ROCK2 protein expression (Western blot assay, mean ± SEM, n = 3). (B) phosphorylation of ROCK2 at Tyr722 (Western blot assay, mean ± SEM, n = 3). (C) ROCK2 activity (ELISA, mean ± SEM, n = 3). * p < 0.05, ** p < 0.01 vs. control group.

Fig 5: ROCK2 Tyr722 mediated NaHS-increased current of BKCa in transfected RHNs (whole−cell patch−clamp recording, mean ± SEM, n = 5). (A) The evoked outward current, and effect of IBTX. (B) Effect of NaHS on BKCa current in the empty plasmid group. (C) Effect of NaHS on BKCa current in the GFP−ROCK2Y722 group. (D) Effect of NaHS on BKCa current in the GFP−ROCK2Y722F group. * p < 0.05, ** p < 0.01 vs. control group. (E) NaHS−increased BKCa currents in each plasmid−transfected RHNs. * p < 0.05, ** p < 0.01 vs. GFP−ROCK2Y722 group. ## p < 0.01 vs. empty plasmid group.