Figure 4. hnRNPA1 and POT1 suppress the accumulation of RPA at telomeres.

a, RPA-coated ssTEL was incubated with WCEs from cells in G1, early S, late S, and M phases of the cell cycle (see Supplemental Methods). The remaining RPA2 on ssTEL was analyzed after incubation. Cyclin A and phospho-histone H3 serve as cell-cycle markers, and histone H4 as a loading control. b, TERRA was analyzed by RNA FISH in HeLa cells following thymidine release. TRF2 severs as a marker of telomeres. c, HeLa cells were treated with hnRNPA1 siRNA or mock treated, and then immunostained with antibodies to RPA2 and TRF2 (left panel). The cells with RPA foci (>5) were quantified (right panel). Mean+s.d., n=3 for mock and sihnRNPA1-1, n=2 for sihnRNPA1-2. d, Chromatin immunoprecipitation of RPA was performed with two different RPA2 antibodies. The association of RPA with telomeres was analyzed by dot blot using a telomere probe and quantified. e, HeLa cells transfected with POT1 or LacZ siRNA were released from a thymidine block. At the indicated times, the G2/M population was determined by FACS (see Fig. S10c). Cells were immunostained for RPA2 and TRF2 (left panel). The cells with RPA foci (>5) were quantified (right panel). Mean+s.d., n=3.