RB activation in irradiated or palbociclib treated cells. (A) Percentage of foci classified into different categories of skeleton dot lengths in IMR-90 cells from the experiment described in Fig. 3 D. Highlighted rows show time points at which a major increase in dispersion was observed. CTRL, control. (B) The changes in RB phosphorylation during IR-induced senescence in IMR-90 cells. Western blots show a major and prolonged loss of RB phosphorylation at all the tested sites, observed first 48 h after IR treatment and through the time course for IR-treated cells. Nontreated cells show reduction in some RB phosphorylation forms at later time points (192 and 288 h) owing to contact inhibition–induced G1 arrest but retain overall phosphorylation. MW, molecular weight. (C) β-Galactosidase staining of the same populations of cells. Scale bar = 190 µm. (D) Changes in expression of key IR-induced senescence signature genes over the time course in IMR-90 cells. Four genes, ICAM1, CCND1, LMNB1 and HDDC2, show dynamic changes as the cells progress toward senescence. Graphs show fold-changes (enrichment over 0-h time point) for the four genes in CTRL (untreated) and IR-treated samples. Three technical replicates per sample were used to calculate fold-changes. (E) Percentage of G1 cells for WT RPE contact-inhibited cells, ΔCDK-RB cells, and both treated with palbociclib for 72 h. (F) Western blot analysis of the cells in E with the indicated antibodies. Note the presence of only the unphosphorylated RB form (lower molecular weight band) in ΔCDK-RB, palbociclib-treated WT, and ΔCDK-RB samples. Source data are available for this figure: SourceData FS1.
Figure S1.

RB activation in irradiated or palbociclib treated cells. (A) Percentage of foci classified into different categories of skeleton dot lengths in IMR-90 cells from the experiment described in Fig. 3 D. Highlighted rows show time points at which a major increase in dispersion was observed. CTRL, control. (B) The changes in RB phosphorylation during IR-induced senescence in IMR-90 cells. Western blots show a major and prolonged loss of RB phosphorylation at all the tested sites, observed first 48 h after IR treatment and through the time course for IR-treated cells. Nontreated cells show reduction in some RB phosphorylation forms at later time points (192 and 288 h) owing to contact inhibition–induced G1 arrest but retain overall phosphorylation. MW, molecular weight. (C) β-Galactosidase staining of the same populations of cells. Scale bar = 190 µm. (D) Changes in expression of key IR-induced senescence signature genes over the time course in IMR-90 cells. Four genes, ICAM1, CCND1, LMNB1 and HDDC2, show dynamic changes as the cells progress toward senescence. Graphs show fold-changes (enrichment over 0-h time point) for the four genes in CTRL (untreated) and IR-treated samples. Three technical replicates per sample were used to calculate fold-changes. (E) Percentage of G1 cells for WT RPE contact-inhibited cells, ΔCDK-RB cells, and both treated with palbociclib for 72 h. (F) Western blot analysis of the cells in E with the indicated antibodies. Note the presence of only the unphosphorylated RB form (lower molecular weight band) in ΔCDK-RB, palbociclib-treated WT, and ΔCDK-RB samples. Source data are available for this figure: SourceData FS1.

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