Fig. 6: RPA condensation is linked to telomere maintenance.
a, RPA2-depleted U-2 OS cells stably expressing WT GFP-RPA2 (encoded by an siRNA-resistant sequence) or the S/T→D mutant were stained for PML and TRF2 to quantify APBs. Averages and s.d. from n = 3 independent samples, 50 cells per replicate. Two-way ANOVA with Šidák`s test, control **P = 0.0047, APH **P = 0.0077. b, Cells treated as in a were EdU labeled for cell cycle staging by QIBC, and G2 cells were analyzed for EdU-positive TRF2 foci. Averages and s.d. from n = 3 independent samples for 100 G2 cells per replicate. Two-way ANOVA with Šidák`s test, control ****P < 0.0001, APH **P = 0.0024. c, Cells were treated the same as in b, but G1 cells were analyzed. Averages and s.d. from n = 3 independent samples for 100 G1 cells per replicate. Two-way ANOVA with Šidák`s test, control ***P = 0.0002, APH ****P < 0.0001. d, Cells were treated as in a for non-denaturing TelG-FISH. Averages and s.d. from n = 4 independent samples (>1,000 cells per sample). Two-way ANOVA with Šidák`s test, control ***P = 0.0004, APH ****P < 0.0001. e, Cells were treated as in a for C-circle analysis by quantitative PCR. Averages and s.d. from n = 3 replicates. Two-way ANOVA with Šidák`s test, control n.s. P = 0.1691, APH ***P = 0.0002. f, Cells were treated as in a for RAD52 foci analysis. Averages and s.d. from n = 3 independent samples (>2,000 cells per sample). Two-way ANOVA with Šidák`s test, control n.s. P = 0.2346, APH **P = 0.0066. g, Cells were treated as in a for TRF2-RAD52 co-localization analysis. Averages and s.d. from n = 3 independent samples (>75 cells per sample). Two-way ANOVA with Šidák`s test, control n.s. P = 0.5668, APH **P = 0.0032. h, Cells were treated as in a for RPA foci fusion analysis from n = 3 independent 48-hour time-lapse experiments, 100 cells per replicate. Averages and s.d. are shown. Two-way ANOVA with Šidák`s test, control n.s. P = 0.8233, APH *P = 0.0170. i, Telomere loss in U-2 OS cells expressing GFP-RPA2 WT or S/T→D by metaphase telomere FISH analysis. Averages and s.d. from n = 3 independent samples (chromosomes/metaphases: WT n1 = 843/16, n2 = 952/16, n3 = 580/11; S/T→D n1 = 830/14, n2 = 949/16, n3 = 378/8). Two-tailed unpaired t-test, ***P = 0.001. Scale bar, 1 µm. j, Model of RPA condensate formation by ssDNA-seeded RPA self-assembly.
