Fig. 3
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)