Fig 2: Roles of ROCK and PKC in vasoconstriction of isolated retinal venules to ET-1. (A) Venular diameters were recorded before (R: resting diameter) and after 30-minute treatment (T) with ROCK inhibitor H-1152 (3 µM; n = 6) or PKC inhibitor Gö 6983 (10 µM; n = 7). In the absence of treatment (Control; n = 12), retinal venules constricted to ET-1 in a concentration-dependent manner. The resting diameter of retinal venules was not altered by either drug treatment. The ET-1–induced constriction was abolished by H-1152 but not Gö 6983. *P < 0.05 versus Control. (B) Venular diameters were recorded under resting conditions (R) and at different concentrations of PKC activator PDBu (n = 6). (C) Equal amount of protein was loaded for Western blot analyses of ROCK isoforms (ROCK1 and ROCK2) and p38 in retinal venules (RV) and neural retina tissue (RT). Data represent three independent experiments.

Fig 3: The inhibition of ROCK signaling promotes the cellular senescence of HDFs. (A) HDFs were treated with PBS or 10 µM Y-27632, then were collected at the indicated times, stained with annexin V-FITC combined with PI, then analyzed by FACS for apoptotic cells. (B) HDFs were treated with PBS (upper) or 10 µM Y-27632 (lower), then fixed at the indicated times and analyzed using a SA-ß-gal staining kit for senescent cells. scale bar = 100 µm. (C) Quantification of (B), the percentage of SA-ß-gal positive cells (blue, arrows) was calculated based on counting a total of 500 cells. (D) HDFs were transfected with siRNAs for ROCK1 and/or ROCK2, or with a scrambled siRNA as a control (sictrl), and at 48 h after transfection, cells were fixed and stained with SA-ß-gal for senescent cells (blue, arrows), scale bar = 100 µm. (E) Quantification of SA-ß-gal-positive cells in (D), the percentage of SA-ß-gal-positive cells was calculated based on counting a total of 500 cells. All experiments were performed at least 3 times,*P<0.05, **P<0.01, ***P<0.005 when two groups were compared as indicated or were compared to the corresponding controls.

Fig 4: miR-133b overexpression downregulates RhoA, ROCK1 and LIMK mRNA expression levels. The relative mRNA expression levels of RhoA (A), ROCK1 (B), LIMK (C) and MLC (D) were measured by reverse transcription-quantitative polymerase chain reaction. n=3. *P<0.05 and **P<0.01 vs. normal group. #P<0.05 and ##P<0.01 vs. high glucose group. hRECs, human retinal endothelial cells; miR, microRNA; RhoA, ras homolog family member A; ROCK1, Rho-associated protein kinase 1; LIMK, LIM domain kinase 1; MLC, myosin light chain; NC, negative control; HG, high glucose.

Fig 5: ROCK1 interacts with Rac1b in vitro and in vivo. (A) The AH109 yeast strain was transformed with either the GAL4-BD fusion plasmid pGBKT7 or pGBKT7-ROCK1-M. The pGADT7-Rac1b clone was selected for further study. (B) Co-immunoprecipitation of Flag-ROCK1-M with Myc-Rac1b. (C) Localization of ROCK1 and Rac1b (Postnuclear supernatant (PNS), mitochondrial fraction (Mito-enriched: pellet), or cytosol (SN: supernatant)). (D) Confocal imaging of Rac1b (green) and mitochondria (red; Scale bar = 5 µm).