Supplementary Figure 3: Nuclear pores and INMPs are required for relocalizing heterochromatic DSBs and do not affect euchromatic repair kinetics.
From: Heterochromatic breaks move to the nuclear periphery to continue recombinational repair
(a) Top: western blots show Nup153 and Mtor RNAi efficiencies. Lamin and a non-specific band (∗) are loading controls. Middle: IF analyses and quantitations show Nup107 or Koi RNAi efficiencies. Bottom: qPCR analyses show Nup160, Nup205, Nup93, Nup50, or Spag4 RNAi efficiencies. (b) Top: IF analyses show the effect of Nup153, Nup107 or Nup160 RNAi depletion on nuclear pore stability (FG-porin signal, labeled with MAb414 antibody) and Nup107 association with the nuclear periphery. Nup153 RNAi effects were independently confirmed with a second set of siRNAs (not shown). Bottom: IF analysis shows no effects of Mtor, Nup50, Nup205 or Nup93 RNAi on Nup107 recruitment to the nuclear periphery. (c) Quantitation of cells fixed before and after IR and processed for IF shows the number of γH2Av foci in DAPI-bright (left) and DAPI-weak (right) after RNAi depletion of the indicated proteins (error bars: s.e.m., ∗∗∗∗p < 0.0001 for Nup153 versus Ctrl RNAi, two-tailed Mann-Whitney test with n > 87 cells for either 60 min or 4 h and for each RNAi condition, from one experiment). Note: Ctrl RNAi samples are the same as in Fig. 3b, d. (d–h) Quantitations of DAPI-weak foci in the experiments described in Fig. 3b (related to Supplementary Fig. 3d), Fig. 3c (related to Supplementary Fig. 3e), Fig. 3d (related to Supplementary Fig. 3f), Fig. 3e (related to Supplementary Fig. 3g), and Fig. 3f (related to Supplementary Fig. 3h). (i) As in Fig. 3f except that 2.5 or 10 Gy were used to induced DSBs (p < 0.05 for all comparisons versus Ctrl at either 2.5 Gy or 10 Gy, n > 140 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). Ctrl/C = control. Scale bars = 1 μm. Images are middle Z-stacks of nuclei. Exact n values are in Supplementary Table 1.
