Rev1, Rev3, or Rev7 siRNA Abolishes Ultraviolet Light-Induced Translesion Replication in HeLa Cells: A Comprehensive Study Using Alkaline Sucrose Density Gradient Sedimentation
Figure 5
Efficient knockdown by Polι siRNAs and the effects on UV-induced TLS in HeLa cells (ASDG profiles of replication products). (a) Efficiency of knockdown on Polι expression RT-PCR analysis and western blot analysis); (b) Effects of two Polι siRNAs on UV-TLS. Twenty-four hours after Polι siRNA transfection, total RNA was isolated and Polι RNA was quantified by RT-PCR. Results were shown in MultiNA gel images and the expression level was presented under the panel (a). Forty hours after Polι siRNA transfection, whole cell extracts were prepared and Polι protein was quantified by western blot analysis (a). Forty hours after Polι siRNA transfection, cells were UV-irradiated (10 J/m2), incubated in normal medium for 30 minutes, pulse-labelled with 10 μCi/mL of [14C]thymidine for 1 hour, then washed twice with PBS, and incubated for 5 hours at 37°C in normal medium (b). Sedimentation is from right to left. The arrow indicates the position of T4 phage DNA (166 kb, i.e., approximately 5.5 × 107 Da/single strand). Average fragment length (in Mb) of each profile is shown in square brackets.
