19q13.42 locus positions closer to nucleoli in ∆CDK-RB cells. Autophagy response in mPRB-252 and WT-RB expressing cells. (A) Quantitation of the shortest distance between the nearest nucleoli and 19q13.42 FISH signal in WT and ΔCDK-RB cells and coordinates of nucleolar associated domains (NADs) mapped to the region (Dillinger et al., 2017). Numbers of foci quantified for each sample (n) are as follows (in the order they appear on the bar graphs): n = 11, 23. Nonparametric two-tailed Mann–Whitney U test was performed for pairs of sample on the graph. Chr., chromosome. (B) Representative LC3B and LAMP2 IF staining images for mP-RB 252 cells 24, 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. (C) Representative LC3B and LAMP2 IF images for WT 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. (D) qPCR of four genes (ATG4B, PSAP, VCP, and SEC23B), 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. For D, asterisks denote P values (comparison with 24-h time point samples) from one-way ANOVA and Newman–Keuls multiple comparison test. ns, P > 0.05; **, P ≤ 0.01.