A Bacterial Cytotoxin Identifies the RhoA Exchange Factor Net1 as a Key Effector in the Response to DNA Damage (original) (raw)
Figure 2
Net1 knock down prevents RhoA activation upon induction of DNA damage.
HeLa cells were transfected with plasmids expressing the control or Net1 specific shRNAs (panel A), or control or Net1 specific siRNA (panel B). Expression of the endogenous Net1 was analysed by western blot 96h (A) or 72h (B) after transfection. Percentage inhibition was calculated as (1- residual Net1)×100, where residual Net1 is defined as the ratio between the optical density of the Net1 specific band in cells transfected with the Net1 iRNA or control iRNA and the optical density of the Net1 specific band in the non-transfected cells. C) HeLa cells, transfected with control or Net1 specific shRNA or shRNA, were left untreated, or exposed to IR (20 Gy), CDT (2 μg ml−1), or CNF-1 (1 μg ml−1), respectively, and further incubated for 4h. Activation of RhoA was assessed by RhoA specific G-LISA™ (mean ±SD of 5 independent experiments for IR and CNF, mean±SD of 3 independent experiments for CDT). Since the effects of transfection with specific Net1 shRNA or siRNA were similar, the data from all these experiments have been summarized together and indicated as iRNA. The fold increase represents the ratio between the levels of GTP-bound RhoA in treated cells and the levels of GTP-bound RhoA in untreated cells. According to the paired t test, the reduced RhoA activation in irradiated or intoxicated cells transfected with Net1 iRNA is statistically significant, while the effect of iRNA on the CNF1-induced RhoA activation is not statistically significant. D) HeLa cells non-transfected or transfected with control or Net1 specific siRNA were exposed to CNF1 (1 μg ml−1) for 6h. The actin cytoskeleton was visualized by TRITC-phalloidin staining. The values represent the percentage of cells with stress fibers. Cells exhibiting more than 5 stress fibers were scored as positive.