Figure 7: MAGE-A4 promotes TLS and DNA-damage tolerance.

(a) H1299 cells were transiently transfected with MAGE-A4 or non-targeting siRNAs. After 16 h, cells were transfected with a siRNA-resistant MAGE-A4 expression plasmid (or empty vector control). Forty-eight hours later, cells were sham or ultraviolet irradiated (20 J m⁻²) and harvested for immunoblot analysis after 2 h. (b) H1299 cells were transfected with siRNA against RAD18, MAGE-A4 or non-targeting siRNA. Forty-eight hours later, cells were pulsed labelled with BrdU (10 μM) for 1 h and collected for flow cytometry. (c) H1299, A549 or mouse embryonic fibroblast (MEF) cells were transfected with a MAGE-A4 expression plasmid or empty vector. After 48 h, cells were sham or ultraviolet irradiated (20 J m⁻²) and extracted 2 h later for immunoblotting. Human dermal fibroblasts (HDFs) stably transduced with a pINDUCER-MAGE-A4 were treated with indicated doxycycline concentrations for 48 h and then collected for immunoblotting. (d) H1299 cells were transfected with siRNA against RAD18 and MAGE-A4 (or with non-targeting oligonucleotides). Twenty-four hours post transfection, cells were re-plated in 24-well dishes and ultraviolet irradiated (5 J m⁻²) 48 h later. DNA synthesis rates were measured immediately before and at different times after ultraviolet treatment. (e) H1299 cells were transfected with siRNA against MAGE-A4 or with non-targeting siRNA. Seventy-two hours post transfection, cells were sham or ultraviolet irradiated (5 J m⁻²) and harvested at different times for immunoblotting. (f) 293T cells were co-transfected with ultraviolet-damaged pSP189 reporter plasmid and MAGE-A4 or RAD18 expression vectors. Forty-eight hours later, 293T cell extracts were collected for immunoblot analysis of MAGE-A4 and RAD18 (right). Recovered pSP189 plasmid was transformed into electro-competent MBM7070 bacteria and pSP189 mutation rates were determined by enumerating blue and white bacterial colonies. Data represent means±s.e.m. of four independent experiments each performed in triplicate. P-values were calculated using a two-tailed Student’s t-test. Baseline mutation rates for the experiments ranged from 5.6 to 9.6%.