Rev1, Rev3, or Rev7 siRNA Abolishes Ultraviolet Light-Induced Translesion Replication in HeLa Cells: A Comprehensive Study Using Alkaline Sucrose Density Gradient Sedimentation
Figure 1
Efficient knockdown by Rev3 siRNAs and their effects on UV-induced TLS in HeLa cells (ASDG profiles of replication products). (a) Efficiency of knockdown on Rev3 expression (RT-PCR analysis); (b) Effects of various Rev3 siRNAs; (c) Effects of Rev3 control siRNAs; (d) Dose-response of siRev3-D; (e) Effect of various Rev3 siRNAs (no UV control). Twenty-four hours after Rev3 siRNA transfection, total RNA was isolated and Rev3 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 Rev3 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, washed twice with PBS, and incubated for 5 hours at 37°C in normal medium (b, c, d). siRev3cont-A, 6 nt mismatches; siRev3cont-B, 4 nt mismatches; siRev3cont-C, 2 nt mismatches (c). Forty hours after Rev3 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 (e). Some of these profiles overlap (c, d, e). 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). Labelled E.coli DNA (approximately 4 Mb) sedimented near the bottom (fractions 3–6) (4). Average fragment length (in Mb) of each profile is shown in square brackets as fragment length of the median fraction.
