Investigation of the role of DNA damage, WAPL, and CAPD3 in RB-mediated dispersion. RB activation increases chromatin resistance to MNase digestion. (A) PLA images for WT and ΔCDK-RB after 24 and 48 h of DOX induction. Just 24 h of ΔCDK-RB expression leads to higher RB-TOPIIβ PLA interaction foci, when compared with WT cells. A similar increase was observed after 48 h of ΔCDK-RB induction. Images show G1 cells. (B) Average numbers of RB-TOPIIβ PLA foci per G1 cell were higher for ΔCDK-RB after 24 and 48 h of DOX induction, when compared with WT. Negative controls (using only RB or TOPIIβ antibody for PLA assay) showed low average numbers of PLA foci in both WT and ΔCDK-RB cells, implying that there was very little background signal. Numbers of foci quantified for each sample (n) are as follows: n = 95 (WT, RB-TOPIIβ 24 h), 214 (ΔCDK-RB, RB-TOPIIβ 24 h), 29 (WT, RB 24 h), 83 (ΔCDK-RB, RB 24 h), 70 (WT, TOPIIβ 24 h), 74 (ΔCDK-RB, TOPIIβ 24 h), 80 (WT, RB-TOPIIβ 48 h), 162 (ΔCDK-RB, RB-TOPIIβ 48 h), 69 (WT, RB 48 h), 110 (ΔCDK-RB, RB 48 h), 70 (WT, TOPIIβ 48 h), 130 (ΔCDK-RB, TOPIIβ 48 h). Error bars show SEM. Nonparametric two-tailed Mann–Whitney U test was performed for pairs of samples indicated on the graph, and asterisks denote P values. (C) Mean skeleton dot lengths (chromosome 7 α-satellite probe) after WT and ΔCDK-RB RPE cells were treated with IR or camptothecin (CPT) to induce DNA damage. Numbers of foci quantified for each sample (n) are as follows (in the order they appear on the bar graphs): n = 21, 28, 25, 26, 21, 24. Error bars are SEM. One-way ANOVA and Holm–Sidak multiple comparison tests were performed, and asterisks denote P values. (D) Western blot analysis of cells in C show that IR and CPT induced H2A.X Ser139 phosphorylation in both WT and ΔCDK-RB–expressing cells. Note that IR- or CPT-induced DNA damage does not affect the ΔCDK-RB–induced chromatin dispersion. MW, molecular weight. (E, G, and I) Mean skeleton dot lengths (chromosome 7 α-satellite probe) after WT and ΔCDK-RB RPE cells were treated with control and CAPD3 siRNAs (E), control and WAPL siRNAs (G), or DMSO and DRB (I). Note that none of these treatments significantly modified dispersion levels in either WT or ΔCDK-RB–expressing RPE cells. Numbers of foci quantified for each sample (n) are as follows (in the order they appear on the bar graphs): E, n = 40, 41, 36, 39; G, n = 40, 47, 36, 34; I, n = 29, 51, 58, 53. Error bars are SEM. For E, G, and I, nonparametric two-tailed Mann–Whitney U test was performed for pairs of samples indicated on the graphs, and asterisks denote P values. ns, P > 0.05; *, P ≤ 0.05; ****, P ≤ 0.0001. (F and H) Western blots for WT and ΔCDK-RB cells treated with control and WAPL siRNAs (F) and control and CAPD3 siRNAs (H). (J) MNase digestion profile of WT and ΔCDK-RB samples. Left: MNase digestion profile for WT and ΔCDK-RB nuclei treated with 40 gel units of MNase per reaction. Right: MNase digestion profile for WT and ΔCDK-RB cells treated with 1,500 units of MNase per reaction. All other conditions were the same. Note: WT samples resolve into a ladder-like typical MNase digestion pattern as early as 30 s at higher MNase concentrations and 1 min at lower concentrations. The ΔCDK-RB sample does not digest into a ladder-like pattern at 30 s at higher MNase concentrations and at even 1 min at lower concentrations (red arrows). A clear MNase digestion ladder is seen at 3 min (left gel) and 2 min (right gel) in the ΔCDK-RB samples, much later than the laddering in WT cells. 1 µg DNA was loaded per well. DNA samples were run on 0.8% agarose gel. Dashed lines indicate the cutoffs for defining the categories compact, diffused, and dispersed for chromosome 7 α-satellite. Source data are available for this figure: SourceData FS4.
Figure S4.

Investigation of the role of DNA damage, WAPL, and CAPD3 in RB - mediated dispersion. RB activation increases chromatin resistance to MNase digestion. (A) PLA images for WT and ΔCDK-RB after 24 and 48 h of DOX induction. Just 24 h of ΔCDK-RB expression leads to higher RB-TOPIIβ PLA interaction foci, when compared with WT cells. A similar increase was observed after 48 h of ΔCDK-RB induction. Images show G1 cells. (B) Average numbers of RB-TOPIIβ PLA foci per G1 cell were higher for ΔCDK-RB after 24 and 48 h of DOX induction, when compared with WT. Negative controls (using only RB or TOPIIβ antibody for PLA assay) showed low average numbers of PLA foci in both WT and ΔCDK-RB cells, implying that there was very little background signal. Numbers of foci quantified for each sample (n) are as follows: n = 95 (WT, RB-TOPIIβ 24 h), 214 (ΔCDK-RB, RB-TOPIIβ 24 h), 29 (WT, RB 24 h), 83 (ΔCDK-RB, RB 24 h), 70 (WT, TOPIIβ 24 h), 74 (ΔCDK-RB, TOPIIβ 24 h), 80 (WT, RB-TOPIIβ 48 h), 162 (ΔCDK-RB, RB-TOPIIβ 48 h), 69 (WT, RB 48 h), 110 (ΔCDK-RB, RB 48 h), 70 (WT, TOPIIβ 48 h), 130 (ΔCDK-RB, TOPIIβ 48 h). Error bars show SEM. Nonparametric two-tailed Mann–Whitney U test was performed for pairs of samples indicated on the graph, and asterisks denote P values. (C) Mean skeleton dot lengths (chromosome 7 α-satellite probe) after WT and ΔCDK-RB RPE cells were treated with IR or camptothecin (CPT) to induce DNA damage. Numbers of foci quantified for each sample (n) are as follows (in the order they appear on the bar graphs): n = 21, 28, 25, 26, 21, 24. Error bars are SEM. One-way ANOVA and Holm–Sidak multiple comparison tests were performed, and asterisks denote P values. (D) Western blot analysis of cells in C show that IR and CPT induced H2A.X Ser139 phosphorylation in both WT and ΔCDK-RB–expressing cells. Note that IR- or CPT-induced DNA damage does not affect the ΔCDK-RB–induced chromatin dispersion. MW, molecular weight. (E, G, and I) Mean skeleton dot lengths (chromosome 7 α-satellite probe) after WT and ΔCDK-RB RPE cells were treated with control and CAPD3 siRNAs (E), control and WAPL siRNAs (G), or DMSO and DRB (I). Note that none of these treatments significantly modified dispersion levels in either WT or ΔCDK-RB–expressing RPE cells. Numbers of foci quantified for each sample (n) are as follows (in the order they appear on the bar graphs): E, n = 40, 41, 36, 39; G, n = 40, 47, 36, 34; I, n = 29, 51, 58, 53. Error bars are SEM. For E, G, and I, nonparametric two-tailed Mann–Whitney U test was performed for pairs of samples indicated on the graphs, and asterisks denote P values. ns, P > 0.05; *, P ≤ 0.05; ****, P ≤ 0.0001. (F and H) Western blots for WT and ΔCDK-RB cells treated with control and WAPL siRNAs (F) and control and CAPD3 siRNAs (H). (J) MNase digestion profile of WT and ΔCDK-RB samples. Left: MNase digestion profile for WT and ΔCDK-RB nuclei treated with 40 gel units of MNase per reaction. Right: MNase digestion profile for WT and ΔCDK-RB cells treated with 1,500 units of MNase per reaction. All other conditions were the same. Note: WT samples resolve into a ladder-like typical MNase digestion pattern as early as 30 s at higher MNase concentrations and 1 min at lower concentrations. The ΔCDK-RB sample does not digest into a ladder-like pattern at 30 s at higher MNase concentrations and at even 1 min at lower concentrations (red arrows). A clear MNase digestion ladder is seen at 3 min (left gel) and 2 min (right gel) in the ΔCDK-RB samples, much later than the laddering in WT cells. 1 µg DNA was loaded per well. DNA samples were run on 0.8% agarose gel. Dashed lines indicate the cutoffs for defining the categories compact, diffused, and dispersed for chromosome 7 α-satellite. Source data are available for this figure: SourceData FS4.

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