Fig. 6
Expression of FLAG-tagged RAD51 adversely affects the response of melanoma cells to cisplatin. a No cisplatin-induced reduction in levels of FLAG-RAD51. Western blots from control A375 cells, cells transfected to express FLAG-RAD51 at levels equivalent to endogenous RAD51 and cells of both types treated with 1 μM cisplatin for 24, 48 or 72 h. The blot was probed sequentially for RAD51 (RAD51 37 kDa, FLAG-RAD51 38.2 kDa), the FLAG tag, and β-actin (42 kDa). b Reduced growth in cisplatin-treated cultures expressing FLAG-RAD51. The table shows the mean cell growth (±SEM, n = 4) of wells after 72 h of 1 μM cisplatin treatment for non-transfected A375 cells expressed as a percentage of the untreated control, and for FLAG-RAD51 transfected A375 cells expressed as a percentage of the non-cisplatin-treated FLAG-RAD51 transfection control. The P value for Student’s paired t test, comparing the effect of cisplatin on growth of normal and FLAG-RAD51-expressing cells is also shown. c Increased early apoptosis in cisplatin-treated A375 cultures expressing FLAG-RAD51. Cultures treated as in (a) and western blotted for activated caspase-3 and β-actin after 48 h of 1 μM cisplatin treatment. Note the highest levels of cleaved caspase-3 (at 17 and 19 kDa) in the sample transfected with FLAG-RAD51 and treated with cisplatin. d Increased apoptosis in cisplatin-treated A375 cells expressing FLAG-RAD51. Cultures treated as in (a) after 72 h of 1 μM cisplatin. A representative flow cytometry profile for each culture condition is shown, with the percentage of cells with subG1 (apoptotic), G1, S and G2/M DNA contents indicated across the top of each profile. Note the highest level of subG1 (apoptotic) material in the cisplatin-treated FLAG-RAD51-expressing cells. The table below shows the mean level of apoptosis (±SEM, n = 4) in wells after 72 h of cisplatin treatment. The P values for Student’s paired t test, comparing the level of apoptosis between cisplatin-treated and untreated control wells, and between cisplatin-treated FLAG-RAD51-expressing wells and untreated FLAG-RAD51-expressing wells are also shown