Rev1, Rev3, or Rev7 siRNA Abolishes Ultraviolet Light-Induced Translesion Replication in HeLa Cells: A Comprehensive Study Using Alkaline Sucrose Density Gradient Sedimentation
Figure 3
Efficient knockdown by Rev1 siRNAs and their effects on UV-induced TLS in HeLa cells (ASDG profiles of replication products). (a) Efficiency of knockdown on Rev1 expression RT-PCR analysis and western blot analysis); (b) Effects of various Rev1 siRNAs on UV-TLS; (c) Dose-response of siRev1-C on UV-TLS; (d) Effect of various siRNAs (no UV control). Twenty-four hours after Rev1 siRNA transfection, total RNA was isolated and Rev1 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 Rev1 siRNA transfection, whole cell extracts were prepared and Rev1 protein was quantified by western blot analysis (a). Forty hours after Rev1 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, c). Forty hours after Rev1 siRNA transfection, cells were not UV-irradiated, pulse-labelled with 10 μCi/mL of [14C]thymidine for 30 minutes, washed twice with PBS, and incubated at 37°C in normal medium for 1 hour (d). Some of these profiles overlap (b, c). 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.
