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

Figure 5

Alkaline comet assays showing DNA single- and double-strand break formation and DNA repair kinetics up to 6 h after exposing MeWo, MeWo-RhoA-V14, and MeWo-RhoA-N19 clonal cells to UVA, UVB, or UVC radiation. In these experiments, control (Ctl.) refers to 0 h or non-UV radiation treatment. A total of 2 × 10⁴ cells were plated on 35 mm culture dishes 24 h before the treatment, followed by trypsinization, suspension in low-melting point agarose, and spreading onto glass slides. After 2 h of electrophoresis in an appropriate buffer, the cell nuclei were stained with ethidium bromide (see details in Materials and Methods), as shown in (b), and many different parameters were acquired using a Nikon microscope controlled by Komet 6.0 software (Andor Technology, Oxford, UK). The most relevant parameter, the olive tail moment, was used to quantify DNA damage and repair in the cells under these conditions (bar graphs in (a)). The graphs represent the average and standard deviation of at least three independent experiments, and the statistical significance of the results was obtained by comparing the effects of different treatments between the MeWo cells and the mutant cells using two-way ANOVA. ; ; .