Fig 2: Evaluation of the role of ROCK2 in radiation response in SiHa cells. a Immunoblot analysis of whole cell extracts of SiHa cells showed an upregulation of both ROCK1 and ROCK2 proteins upon irradiation at the indicated time points. b Immunofluorescence analysis also showed that the ROCK2 expression levels were higher in the nuclear compartment as compared to the ROCK1 levels. Scale bar = 10 μm.(n = 3). c Immunoblot analysis of ROCK1 and ROCK2 at different times points in the nuclear extracts of IR SiHa cells (n = 3). d Graphical representation of flow cytometry analysis showing an increased percentage of cells with ROCK2 expression in the irradiated (IR) SiHa cells as compared to the non-irradiated (NR) control cells. A fold increase of 1.4 is depicted (p < 0.03; n = 3). e Inhibition of ROCK signaling using Y27632 (10 μM) to determine its effect on cell survival. There was a 30% increase in cell death of the treated cells due to irradiation (n = 3, *p < 0.037; # n.s). f SiHa cells transfected with 1 μg of pCAG-ROCK2 expression vector and the corresponding empty vector followed by irradiation displayed increased survival. (p < 0.01, n = 4). g-i SiHa cells treated with saponin (Sap+ve) had comparable cell viability as compared to the untreated control (Sap -ve), #- n.s (n = 3). g-ii Immunoblot analysis showed that there is reduction in ROCK2 levels but not ROCK1 upon inhibition with the ROCK2 antibody (ROCK2In). IgG isotype has been used as control (n = 3). g-iii Representative histograms depict the increased PI uptake in cells with ROCK2In as compared to the corresponding isotype control. g-iv Graphical representation of flow cytometry-based analysis of cell survival upon ROCK2In followed by irradiation shows increased sensitization of SiHa cells to irradiation (n = 3, p < 0.001). h-i Graphical representation of weights of tumors formed by NR and IR cells (NR tumor = 0.53 g ±0.28; IR tumor = 0.76 g ±0.32, n = 3, p < 0.02). h-ii Representative images of immunofluorescence-based analysis of xenografts derived from NR and IR tumors showed increased expression of ROCK2 in IR tumor derived sections (scale bar = 50 μm)

Fig 3: ROCK2’s crosstalk with DNA repair machinery in cervical cancer cells. a Immunoblot of irradiated SiHa cells showing expression levels of DNA repair proteins at various time points indicated, as compared to control at 0 h. b-i Representative immunofluorescence images depict the co-expression of ROCK2 and pH2Ax in CaSki cells at 1 h following irradiation (n = 3, scale bar = 10 μm). b-ii Representative immunofluorescent images of irradiated CaSki cells showing a decreased expression of pH2Ax in ROCK2In as compared to IgGIn cells (n = 3, scale bar = 10 μm). b-iii Box plots of the number of pH2Ax foci in IgGIn and ROCK2In cells following irradiation. A median value of 15 foci/cell in IgGIn as opposed to 5 foci/cell in the ROCK2In was observed (n = 3, *p < 0.01). b-iv Immunoblot analysis demonstrated a reduction in pH2Ax levels at 1 h in irradiated ROCK2In as compared to IgGIn cells (n = 3). b-v Immunoprecipitation using ROCK2 antibody shows pull down of pH2Ax at 1 h following irradiation treatment. IgG isotype is used as the control (n = 3). c-i Representative immunofluorescence images showing a decreased expression of RAD50 as compared to IgGIn, in irradiated CaSki cells, with ROCK2In (n = 3, scale bar = 10 μm). c-ii Immunoblot analysis in irradiated SiHa cells confirming reduction in RAD50 levels upon ROCK2In (n = 3). d-i Representative immunofluorescence images showing a decreased expression of MRE11 as compared to IgGIn cells, in irradiated CaSki cells, with ROCK2In (n = 3, scale bar = 10 μm). d-ii Immunoblot analysis of irradiated SiHa cells also showed a reduction in MRE11 levels upon ROCK2In (n = 3)

Fig 4: OA induced stress fiber formation and cell migration in HUVECs through RhoA/ROCK-II pathway. The amounts of active GTP-bound RhoA (RhoA-GTP, rhotekin-RBD), Rac1 (Rac1-GTP, PAK-PBD), Cdc42 (Cdc42-GTP, PAK-PBD) were determined by a pull-down assay, in non-stimulated (control) or OA-stimulated (2.5 μM) HUVECs for 1 min (1′), 3′, 5′, 10′, 20′ or 30’. Total RhoA, Rac1, Cdc42 in total cell extracts were also detected (A). Quantification of active, GTP-bound state was presented as RhoA-GTP (20′) (B) and Cdc42-GTP (3′) (C). HUVECs treated for 20 min with 2.5 μM OA after serum were starved and subsequently stained with rhodamine-phalloidin and with DAPI for nuclei. RhoA induced actin stress fibers were visible (D). VEGFR2 (E) and ROCK-II (F) mRNA expression in HUVECs were stimulated with 2.5 μM OA for 30, 60 min. HUVECs stimulated with DMSO, 2.5 μM OA for 30, 60 min and VEGFA165 for 60 min. Western blotting analysis of VEGFR2, ROCK-II or p-Cofilin and total Cofilin in HUVECs were performed. β-actin expression was presented as an internal control of protein loading. VEGFA165 was acted as a positive control (G). Quantitative data included VEGFR2 (H), ROCK-II (I) and p-Cofilin (J). Data were represented as mean ± SD (n = 3). *p < 0.05; **p < 0.01 vs. control.

Fig 5: Inhibitory effects of IGF-1 on AngII-induced differentiation of cardiac fibroblasts into myofibroblasts.A Adult rat cardiac fibroblasts were treated with AngII (1 µM) or AngII plus IGF-1 (100 ng/mL) for 24 h, and immunocytochemical staining was performed. Representative images of αSMA and DAPI (magnification, ×40, 25 µm) and B quantification of fluorescence intensity. n = 4–7/group. Cells were treated with AngII for 3–24 h. The lysates were then immunoblotted with the indicated antibodies. Western blot analysis (C) and densitometric ratios (D) of ROCK1, ROCK2, and αSMA. n = 4–6. Cells were transfected with 30 nM negative control or 30 nM ROCK2 siRNA for 24 h and then incubated with vehicle or IGF-1 with AngII for 24 h. Western blot analysis (E) and densitometric ratios (F, G). n = 4 each. Knockdown of ROCK2 was performed in cardiac fibroblasts and treated with AngII for 6 h. Western blot analysis (H) and densitometric ratios (I, K). n = 6–7/group. Cardiac fibroblasts were treated with vehicle or the Smad3 inhibitor SIS3 (5 μM) for 40 min and then incubated with vehicle or IGF-1 with AngII for 24 h. Western blot analysis (L) and densitometric ratios (M–O). n = 6/group. Cardiac fibroblasts were transfected with 10 nM negative control or 5 nM Akt1 and Akt2 siRNA for 24 h and then incubated with vehicle or IGF-1 with AngII for 24 h. Western blot analysis (P) and densitometric ratios (Q–S). n = 4–6/group. Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. T Schematic diagram depicting the roles of IGF-1 signaling in modulating AngII-induced cardiac fibrosis.