Dispersion of FISH signals, a time-dependent change that occurs during IR-induced senescence and in palbociclib-treated cells.(A) IR was used to induce senescence in IMR-90 cells. Dispersion was assessed by measuring the skeleton dot lengths of the chromosome 7 α-satellite FISH signal at the indicated time points. CTRL, control. (B and C) Dispersion was observed to increase at 192 h (B) and 288 h (C) after IR treatment (insets show skeletons of representative foci). (D) Distribution of individual skeleton dot lengths of the FISH signals measured in A. The mean skeleton length and the shift up in distribution of individual measurements demonstrate the increase in dispersion at 192 and 288 h after IR. (E) Skeleton dot length measurements of the FISH signal of the chromosome 7 α-satellite probe in WT RPE1 cells treated with palbociclib for 72 h or cells expressing ΔCDK-RB for 72 h. Palbociclib treatment disperses FISH signal in WT RPE to a degree comparable to 72 h of ΔCDK-RB expression. (F) Skeleton dot length measurements in chromosome 7 α-satellite region in RB+/+ (WT) or RB−/− (CRISPR knockout) cells treated with palbociclib for 5 d. Chromatin dispersion is evident only in RB+/+ (WT) cells. (G) Dispersion caused by ΔCDK-RB expression became statistically significant after 72 h and continued to increase to 144 h. (H) The range of FISH signals obtained for shRNA-mediated knockdown of RB1 and cells expressing scrambled shRNAs in RPE1 cells. Quantitation of mean skeleton dot lengths showed no significant difference in dispersion between RB1 shRNA knockdown and scrambled shRNA samples. Numbers of foci quantified for each sample (n) are as follows (in the order they appear on the bar graphs): D, n = 46, 37, 36, 27, 35, 37, 38, 34, 24, 17; E, n = 37, 38, 39; F, n = 48, 47, 30, 42; G, n = 36, 34, 25, 38, 33, 22; H, n = 78, 53. Error bars are SEM. Nonparametric two-tailed Mann–Whitney U tests were performed for pairs of samples indicated on graphs D–H, and asterisks denote P values; ns, P > 0.05; ****, P ≤ 0.0001. Scale bar for B, C, and H (including insets) = 2 µm. Dashed lines indicate the cutoffs for defining the categories compact, diffused, and dispersed for chromosome 7 α-satellite.
Figure 3.

Dispersion of FISH signals, a time-dependent change that occurs during IR-induced senescence and in palbociclib-treated cells. (A) IR was used to induce senescence in IMR-90 cells. Dispersion was assessed by measuring the skeleton dot lengths of the chromosome 7 α-satellite FISH signal at the indicated time points. CTRL, control. (B and C) Dispersion was observed to increase at 192 h (B) and 288 h (C) after IR treatment (insets show skeletons of representative foci). (D) Distribution of individual skeleton dot lengths of the FISH signals measured in A. The mean skeleton length and the shift up in distribution of individual measurements demonstrate the increase in dispersion at 192 and 288 h after IR. (E) Skeleton dot length measurements of the FISH signal of the chromosome 7 α-satellite probe in WT RPE1 cells treated with palbociclib for 72 h or cells expressing ΔCDK-RB for 72 h. Palbociclib treatment disperses FISH signal in WT RPE to a degree comparable to 72 h of ΔCDK-RB expression. (F) Skeleton dot length measurements in chromosome 7 α-satellite region in RB+/+ (WT) or RB−/− (CRISPR knockout) cells treated with palbociclib for 5 d. Chromatin dispersion is evident only in RB+/+ (WT) cells. (G) Dispersion caused by ΔCDK-RB expression became statistically significant after 72 h and continued to increase to 144 h. (H) The range of FISH signals obtained for shRNA-mediated knockdown of RB1 and cells expressing scrambled shRNAs in RPE1 cells. Quantitation of mean skeleton dot lengths showed no significant difference in dispersion between RB1 shRNA knockdown and scrambled shRNA samples. Numbers of foci quantified for each sample (n) are as follows (in the order they appear on the bar graphs): D, n = 46, 37, 36, 27, 35, 37, 38, 34, 24, 17; E, n = 37, 38, 39; F, n = 48, 47, 30, 42; G, n = 36, 34, 25, 38, 33, 22; H, n = 78, 53. Error bars are SEM. Nonparametric two-tailed Mann–Whitney U tests were performed for pairs of samples indicated on graphs D–H, and asterisks denote P values; ns, P > 0.05; ****, P ≤ 0.0001. Scale bar for B, C, and H (including insets) = 2 µm. Dashed lines indicate the cutoffs for defining the categories compact, diffused, and dispersed for chromosome 7 α-satellite.

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