RB-induced dispersion is associated with increased expression of autophagy genes. (A) Number of transcripts that positively (r > 0.6) or negatively (r < −0.6) correlate with heterochromatin and euchromatin dispersion rankings. (B) Left: Biological pathway analysis for correlated genes. Right: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for correlated genes. FDR, false discovery rate. (C) Representative LC3B and LAMP2 IF staining images for ΔCDK-RB, mP-RB 356, and mP-RB 780 cells 24, 72, and 96 h after DOX induction. Scale bar = 25 µm. (D) Quantification of LC3B-II average puncta per cell for ΔCDK-RB, mP-RB 356, mP-RB 252, and mP-RB 780 24, 48, 72, and 96 h after DOX induction. 48-, 72-, and 96-h time point cells were treated with 100 nM bafilomycin A1 for 24 h before fixation. 24-h time point cells were treated with 100 nM bafilomycin A1 for 3 h before fixation. (E) qPCR of four genes (LC3B, p62, ATG14, and WIPI1), at 24-, 48-, and 72-h time points of DOX induction for ΔCDK-RB, mP-RB 356, mP-RB 252, and mP-RB 780 cell lines. Graphs show fold-changes (enrichment over 24-h time point samples) for the four genes. Three technical replicates per sample were used to calculate fold-changes. (F) Representative LC3B and LAMP2 IF images for WT and ΔCDK-RB induced with DOX for 72 h and treated with 0.5 µM etoposide for the final 24 h. Cells were treated with bafilomycin A1 for 24 h before IF fixation. (G) Quantification of LC3B-II average puncta per cell for WT and ΔCDK-RB untreated and treated with 0.5 µM etoposide for 24 h. We observed that etoposide suppresses LC3-II punta in ΔCDK-RB cells. Scale bar = 50 µm. Numbers of foci quantified for each sample (n) are as follows (in the order they appear on the bar graphs): D, n = 18, 22, 27, 18, 30, 18, 28, 20, 20, 28, 28, 20, 24, 20, 26, 24; G, n = 17, 22, 15, 27. For D, asterisks denote P values from nonparametric Kruskal–Wallis test and Dunn’s multiple comparison test (each sample was compared with its 24-h time point). Error bars are SEM. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
Figure 9.

RB-induced dispersion is associated with increased expression of autophagy genes. (A) Number of transcripts that positively (r > 0.6) or negatively (r < −0.6) correlate with heterochromatin and euchromatin dispersion rankings. (B) Left: Biological pathway analysis for correlated genes. Right: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for correlated genes. FDR, false discovery rate. (C) Representative LC3B and LAMP2 IF staining images for ΔCDK-RB, mP-RB 356, and mP-RB 780 cells 24, 72, and 96 h after DOX induction. Scale bar = 25 µm. (D) Quantification of LC3B-II average puncta per cell for ΔCDK-RB, mP-RB 356, mP-RB 252, and mP-RB 780 24, 48, 72, and 96 h after DOX induction. 48-, 72-, and 96-h time point cells were treated with 100 nM bafilomycin A1 for 24 h before fixation. 24-h time point cells were treated with 100 nM bafilomycin A1 for 3 h before fixation. (E) qPCR of four genes (LC3B, p62, ATG14, and WIPI1), at 24-, 48-, and 72-h time points of DOX induction for ΔCDK-RB, mP-RB 356, mP-RB 252, and mP-RB 780 cell lines. Graphs show fold-changes (enrichment over 24-h time point samples) for the four genes. Three technical replicates per sample were used to calculate fold-changes. (F) Representative LC3B and LAMP2 IF images for WT and ΔCDK-RB induced with DOX for 72 h and treated with 0.5 µM etoposide for the final 24 h. Cells were treated with bafilomycin A1 for 24 h before IF fixation. (G) Quantification of LC3B-II average puncta per cell for WT and ΔCDK-RB untreated and treated with 0.5 µM etoposide for 24 h. We observed that etoposide suppresses LC3-II punta in ΔCDK-RB cells. Scale bar = 50 µm. Numbers of foci quantified for each sample (n) are as follows (in the order they appear on the bar graphs): D, n = 18, 22, 27, 18, 30, 18, 28, 20, 20, 28, 28, 20, 24, 20, 26, 24; G, n = 17, 22, 15, 27. For D, asterisks denote P values from nonparametric Kruskal–Wallis test and Dunn’s multiple comparison test (each sample was compared with its 24-h time point). Error bars are SEM. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

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