Inhibition of the RhoA GTPase Activity Increases Sensitivity of Melanoma Cells to UV Radiation Effects

Figure 6

Immunofluorescence analysis for the detection of cyclobutane pyrimidine dimers (CPDs) generated in the MeWo tumor cell line and in the MeWo-RhoA-N19 and MeWo-RhoA-V14 clones after treatment with UV radiation. Coverslips containing an approximately 80% confluent cell monolayer were exposed to UV radiation and collected after 0 (control or non-UV radiation treatment), 6, 24, or 48 h, followed by fixation with 4% paraformaldehyde, permeabilization with 0.5% Triton X-100, and genomic DNA denaturation in the presence of 2 M HCl. The coverslips were incubated for 2 h in an anti-CPD primary antibody (diluted 1 : 200) and then for 1 h at room temperature in an Alexa Fluor 568 secondary antibody (a). Approximately 50 single cells were photographed at 100x magnification and were quantified in sequence using Zen software (Zeiss). The bars represent the average and standard deviation from three independent experiments (b). Two-way ANOVA was performed to compare the mutant clones with the MeWo clone treated according to the same specified conditions. ; ; ; .