Relationship between cell cycle stage, forms of active RB, and dispersion. (A) The range of signals detected by the chromosome 7 α-satellite probe in Fucci RPE-1 cells. Low-level dispersion was observed in G1 cells but not in S/G2/M cells. (B) Scatter plots depicting distribution of skeleton dot length of signals and mean in S/G2/M and G1 cells. (C and D) The level of dispersion induced by ΔCDK-RB was greater than that seen when RPE cells were arrested by serum starvation (Ser. Stv.) for 72 h. The euchromatin probe 19q13.42 was used for FISH. (C) Representative images of FISH foci in each category. (D) Distribution of skeleton dot lengths, means, and percentages of foci in each category. Quantitation includes biological replicates, set up and performed on different days. (E–J) RB monophosphorylation forms differ in the ability to disperse euchromatin and heterochromatin regions. Mean dot lengths were used to classify RB monophosphorylation forms as high dispersers, medium dispersers, or low dispersers. (E–H) Examples of high dispersers and low dispersers detected with the heterochromatin probe (chromosome 7 α-satellite; E and F) or the 19q13.42 euchromatin probe (G and H). (I and J) Mean dot lengths obtained using heterochromatin and euchromatin regions, respectively. The rankings of the various forms of active RB are shown. Quantitation includes biological replicates, set up and performed on different days. Numbers of foci quantified for each sample (n) are as follows (in the order they appear on the bar graphs): B, n = 17, 18; D, n = 83, 64; I, n = 55, 48, 74, 61, 50, 75, 75, 76, 104, 60, 63, 56, 93, 85, 207, 115; J, n = 60, 84, 52, 113, 124, 144, 66, 120, 80, 106, 80, 56, 145, 69, 99, 111. Error bars are SEM. For B and D (left panel), asterisks denote significance from nonparametric two-tailed Mann–Whitney U test. For B (right panel), asterisks denote P values from multiple t tests (without correction for multiple comparisons). For I and J, asterisks denote P values from nonparametric Kruskal–Wallis test and Dunn’s multiple comparison test. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Scale bar (including insets) = 2 µm. Dashed lines indicate the cutoffs for defining the categories compact, diffused, and dispersed for chromosome 7 α-satellite and compact/diffused, dispersed, and scattered for 19q13.42 probe.
Figure 5.

Relationship between cell cycle stage, forms of active RB, and dispersion. (A) The range of signals detected by the chromosome 7 α-satellite probe in Fucci RPE-1 cells. Low-level dispersion was observed in G1 cells but not in S/G2/M cells. (B) Scatter plots depicting distribution of skeleton dot length of signals and mean in S/G2/M and G1 cells. (C and D) The level of dispersion induced by ΔCDK-RB was greater than that seen when RPE cells were arrested by serum starvation (Ser. Stv.) for 72 h. The euchromatin probe 19q13.42 was used for FISH. (C) Representative images of FISH foci in each category. (D) Distribution of skeleton dot lengths, means, and percentages of foci in each category. Quantitation includes biological replicates, set up and performed on different days. (E–J) RB monophosphorylation forms differ in the ability to disperse euchromatin and heterochromatin regions. Mean dot lengths were used to classify RB monophosphorylation forms as high dispersers, medium dispersers, or low dispersers. (E–H) Examples of high dispersers and low dispersers detected with the heterochromatin probe (chromosome 7 α-satellite; E and F) or the 19q13.42 euchromatin probe (G and H). (I and J) Mean dot lengths obtained using heterochromatin and euchromatin regions, respectively. The rankings of the various forms of active RB are shown. Quantitation includes biological replicates, set up and performed on different days. Numbers of foci quantified for each sample (n) are as follows (in the order they appear on the bar graphs): B, n = 17, 18; D, n = 83, 64; I, n = 55, 48, 74, 61, 50, 75, 75, 76, 104, 60, 63, 56, 93, 85, 207, 115; J, n = 60, 84, 52, 113, 124, 144, 66, 120, 80, 106, 80, 56, 145, 69, 99, 111. Error bars are SEM. For B and D (left panel), asterisks denote significance from nonparametric two-tailed Mann–Whitney U test. For B (right panel), asterisks denote P values from multiple t tests (without correction for multiple comparisons). For I and J, asterisks denote P values from nonparametric Kruskal–Wallis test and Dunn’s multiple comparison test. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Scale bar (including insets) = 2 µm. Dashed lines indicate the cutoffs for defining the categories compact, diffused, and dispersed for chromosome 7 α-satellite and compact/diffused, dispersed, and scattered for 19q13.42 probe.

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