Fig 1: ROCK2 transformed ZEB1 to activator via YAP1.A ChIP assay for ATM promoter with YAP1-siRNA, and expression of ATM protein was determined with YAP1-siRNA. B IP assay for YAP1 and ZEB1. C ROCK2 regulated YAP1 expression. D YAP1 expression in 693 glioma patients as determined by CGGA online tools. E Co-expression analysis of YAP1 and ROCK2 mRNA level in subset of recurrent gliomas. F Luciferase activities were determined after transfection with YAP-1 shRNA in U87R, U87R-ROCK2OE, U251R and U251R-ROCK2-OE cells. Luciferase were detected. G ChIP assay of ZEB1 on ATM promotor was determined with transfection of YAP1-shRNA in U87R, U87R-ROCK2OE, U251R and U251R-ROCK2OE cells. H YAP1, ZEB1 and ATM levels in YAP1-KD cells were determined by WB. I, J ATM gene expression was detected by Q-PCR after transfection of YAP-shRNA or YAP-overexpression plasmid in U87R, U87R-ROCK2OE, U251R and U251R-ROCK2OE cells. K, L ROCK2, YAP1, ZEB1 and ATM expressions were determined by WB after transfection of YAP-shRNA or YAP-overexpression plasmid in U87R, U87R-ROCK2OE, U251R and U251R-ROCK2OE cells. M Mechanisms through which ROCK2 mediates TMZ-resistance in MGMTlow glioma. Data are expressed as mean ± SD of three independent experiments. p values determined by ANOVA or two-tailed unpaired t-test. Statistical differences compared with the controls are given as *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.
Fig 2: ROCK2 directly regulated HR via ATM.HR efficiency was determined with supplementary of MRE11, RAD51, BRCA1 in U87R (A), U251R (B), mrU251 (C) and A172R (D) of ROCK2-KD clones cells. E HR efficiency was determined with supplementary of ATM in ROCK2 KD cells. F HR efficiency was determined with supplementary of ATM-tet-KD in ROCK2-OE cells. RAD51 levels at the site-specific DSB, G U87R, H U251R, I mrU251, J A172R. Data are expressed as mean ± SD of three independent experiments. p values determined by ANOVA or two-tailed unpaired t-test. Statistical differences compared with the controls are given as *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.
Fig 3: ROCK2 suppression induced DSB in TMZ-R cells.A ROCK2 protein expression in TMZ-R cells and parental cells. B, C ROCK2 protein level was determined with ROCK2-shRNA (ROCK2-KD). D WB quantification of B and C. E Neutral comet assay was performed, Scale bars, 60 μm. F–I DSB-ChIP quantification over time of γH2AX level at the site-specific DSB. J γH2AX protein level in ROCK2-KD cells with TMZ (100 μM) for 24 h. K IF results of γH2AX-foci in U87R and U251R with ROCK2-KD. Data are mean ± SD of three independent experiments. p values determined by ANOVA or two-tailed unpaired t-test. Statistical differences compared with the controls are given as *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.
Fig 4: NaHS-induced phosphorylation of ROCK2 or GFP-ROCK2 at Tyr722 in the transfected RHNs. (A) Representative images of transfection. ((a): Empty plasmid-transfected RHNs; (b): ROCK2Y722-pEGFP-N1 plasmid-transfected RHNs; (c): ROCK2Y722F-pEGFP-N1 plasmid-transfected RHNs.) (GFP-tagged, 100 μm). (B) NaHS-promoted phosphorylation of ROCK2 at Tyr722 ((a): empty plasmid; (b): GFP-ROCK2Y722 group; (c): GFP-ROCK2Y722F group) (Western blot assay, mean ± SEM, n = 3). * p < 0.05, ** p < 0.01 vs. control group (p-ROCK2Y722/ROCK2), # p < 0.05, ## p < 0.01 vs. control group (GFP-p-ROCK2Y722/GFP-ROCK2Y722).
Fig 5: ROCK2 Tyr722-mediated protection of NaHS on H/R injury in transfected RHNs (mean ± SEM, n = 6). (A) Cell viability. (B) Release of LDH. (C) Release of NSE. * p < 0.05, ** p < 0.01 vs. H/R group. (D) Inhibitory effects on the decreased cell viability and released LDH and NSE. * p < 0.05, ** p < 0.01 vs. GFP-ROCK2Y722 group.
Supplier Page from Abcam for Anti-ROCK2 (phospho Y722) antibody