Effect of TRIM29 silencing on the cell survival after a single exposure to UVB.
A: Western blot determined the abundance of TRIM29 in TRIM29 shRNA cell lines (3, 3b and 4), in N-hTERT parental cells and in the Neg shRNA cell line at 16 h after a single exposure to UVB at 1,200 mJ/cm2. α-tubulin was used to assess protein loading. B: Cell viability was assessed by the MTT method at 16 h after the exposure to UVB. The abundance of TRIM29 in N-hTERT cells non-exposed to UVB (CTL) was considered as reference level. Results presented are means ± S.D. (N = 4, Student's t-test: NS: non significant, * p<0.05, ** p<0.01 vs UVB in N-hTERT keratinocytes). C: Cleavage of PARP and protein abundance of p53 in TRIM29 shRNA cell line 3, in N-hTERT parental cells and in the Neg shRNA cell line were analysed by Western blotting. The full length PARP protein (116 kDa) and the fragment resulting from PARP cleavage (85 kDa) are indicated. N-hTERT cells exposed to a lethal dose of 2,400 mJ/cm2 of UVB were used as positive control. D: Phosphorylation of ATM in TRIM29 shRNA cell line 3, in N-hTERT parental cells and in the Neg shRNA cell line were analysed by Western blotting with nuclear extracts. Total ATM abundance was used to assess protein loading. E–F: The relative abundance of p21WAF-1 (E) and involucrin mRNA (F) was analysed by RT-PCR at 16 h after the exposure to UVB. The mRNA abundance in N-hTERT keratinocytes not exposed to UVB (CTL) was considered as the 100% reference. The results are expressed as means ± SD (N = 3). NS: non significant, * p<0.05, ** p<0.01 and *** p<0.001 vs N-hTERT CTL cells except that straight lines indicate statistical tests on the ratios obtained between cells exposed to UVB, for each cell condition (N-hTERT, Neg shRNA, TRIM29 shRNA).