Research Paper Volume 16, Issue 20 pp 12977—13011

Figure 2. WRN and HDAC2 proximity is facilitated by HP1α. (A) Clonal isolates of CRISPR/Cas9 knockouts of WRN were generated in the GM639 background. WT1 and WT2 are isogenic WRN wild type controls. WT1 (GM639-Cas9-EV [26]), parent cell line is a GM639 derivative with Cas9 and an empty vector instead of a dgRNA expressing construct. WT2 is one of WRN wild type isolates derived from subcloning of WRN dgRNA-transfected GM639-Cas9 cells. (B) Western blot analysis of the WRN k.o. #3 cell line (hereafter called Δwrn-3) stably transfected with pLX209-neo-WRN expressing 5xmyc-WRN. (C) Δwrn-3 was transfected with non-targeting (siControl) and the HP1α gene-targeting (CBX5-5) siRNAs and analyzed by Western blotting at 36 hrs post-transfection. (D) WI38hTERT expressing non-targeting shNS were transfected with non-targeting and HP1α-targeting (CBX5-5) siRNAs and analyzed by IF in situ with HP1α antibody at 36 hrs post-transfection. (E) Quantitation of V5/HDAC2 PLA performed in hdac2-27 cells stably transfected with HDAC2-V5 transgene or with empty vector (e.v., no tag). Note that not all transfected cells express HDAC2-V5, thus cells with no PLA signal are expected in the hdac2-27/HDAC2-V5 population. (F) Quantitations of HDAC2/HP1α PLA performed in Δwrn-3 and hdac2-27 (left panel), in Δwrn-3 transfected with non-targeting or CBX5-5 siRNAs (center panel), or in Δwrn-3 with or without expression of the WRN transgene (right panel). The graph represents two independent experiments. (G) Quantitations of WRN/HP1α PLA performed in WI38hTERT expressing non-targeting shRNA or shRNA against WRN (left panel), in WI38hTERT expressing non-targeting shRNA and also transfected with siRNA against HP1α or a non-targeting control (center panel, the experiment was done at 36 hrs post-transfection); and in hdac2-27 stably expressing the indicated transgenes or an empty vector (right panel, the graph represents three independent experiments). (H) Quantitations of WRN/HDAC2 PLA performed in WI38hTERT with siRNA against HP1α or a non-targeting control (left panel) and in WT1 (GM639-Cas9-EV) transfected with the same siRNAs (right panel). Crossbars in graphs are distribution means.