Supplementary Figure 2: STUbL and RENi do not affect Smc5/6 association with heterochromatin and repair foci, and euchromatic repair.

(a) Western analysis of cells expressing GFP-Dgrn shows Dgrn RNAi efficiency, and that dRad60 RNAi does not affect Dgrn protein level. (^∗) A non-specific band is the loading control. (b) Western analysis of cells expressing FHA-dRad60 shows dRad60 RNAi efficiency, and that Dgrn RNAi does not affect dRad60 protein level. Actin is the loading control. (c) Quantitation of the experiment described in Fig. 2b shows the number of γH2Av foci in DAPI-weak after RNAi depletions. (d) As in Fig. 2b except that 2.5 or 10 Gy were used to induce DSBs (p < 0.05 for all comparisons versus Ctrl at either 2.5 Gy or 10 Gy, n > 120 cells; error bars represent mean ± s.d. from three independent experiments, whereas the sample size used to determine P values is the total number of cells for each RNAi and dose, pooled across the three experiments). (e) Images (left) and quantitation (right) of Nse2 or Smc6 signals in cells described in Supplementary Fig. 1j show the effects of dRad60 or Dgrn RNAi on Nse2/Smc6 colocalization with the HP1a domain before IR, or Nse2/Smc6 recruitment to damage foci 10 min after IR. Images are maximum intensity projections of nuclei. Scale bars = 1 μm. Error bars: s.e.m.; n = 17 cells/RNAi condition from one experiment. (f) qPCR analysis shows dRad60 RNAi efficiency in the experiment described in g. (g) Ip of GFP-Dgrn was done as in Fig. 2d, except that dRad60 or a control was depleted before Ip. (h) Quantitation of the experiment described in Fig. 2e shows the number of foci in DAPI-weak. Because the number of Rad51 foci in DAPI-bright is a small fraction of the total number of foci, the number of foci in DAPI-weak is similar to the total number of foci (not shown). Ctrl/C = control.