Fig 2: Elevated c-MYC expression prevents T/CQ-mediated cell cycle arrest. (A) Representative cell cycle histograms with percentages of cells in G0/G1, S, and G2/M phases are shown for Panc 10.05, HPAF-II, and PDX220 cells expressing Tet-On c-MYC. Cells were treated for 7 d with DMSO (Ctrl), 2 μg/ml Dox, trametinib (T, at 5–100 nM) and CQ (at 5–20 μM; T/CQ), or D/T/CQ. (B) Average percentage of cells in S phase for Tet-On c-MYC–expressing Panc 10.05, HPAF-II, and PDX220 cells that were treated as above (n = 3 from two independent experiments for each cell type, mean ± SEM). *, P < 0.05; **, P < 0.01; Student’s two-sided t test. (C) Immunoblot analysis of lysates generated from Tet-On c-MYC–expressing cells that were treated as above (n = 1 for each cell type). Lysates were blotted for c-MYC, pERK1/2 (phospho-T202/Y204), ERK1/2, pRB (phospho-S807/S811), RB, E2F1, CCNB1, CCNA2, CCND2, CCNE1, CDK2, CDK4, CDK6, p27KIP1, p21CIP1, and ACTB. Densitometry quantitation of c-MYC and pRB protein levels normalized to actin are shown. Source data are available for this figure: SourceData F3.

Fig 3: Elevated c-MYC expression does not prevent P/CQ-mediated cell cycle arrest. (A) Panc 10.05, PDX220, and MIA-PaCa2 cells were imaged to assess confluence over time using the IncuCyte Live-Cell Analysis System after treatment with palbociclib (P), CQ, or the combination of both agents (P/CQ). Data represent mean values, and error bars reflect SD of one experiment (n = 3). One-way ANOVA was conducted to determine statistical difference between the single agent controls (CDK4/6 inhibitor or CQ) and the combination group. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) MIA-PaCa2 cells were treated with ribociclib (Ribo) or abemaciclib (Abema) alone or in combination with CQ and confluence was measured over time. A one-way ANOVA was utilized to determine statistical significance between the single agent controls and the combination group. The Ribo/CQ experiment was conducted twice (n = 2) and the Abema/CQ experiment was conducted once (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Representative images of MIA-PaCa2 cells shown by phase contrast (top) and the corresponding cell mask utilized to estimate well confluence (bottom) for each treatment at 48 h. Bars are 300 μm. (D) Average percentage of cells in S phase for Tet-On c-MYC–expressing Panc 10.05, HPAF-II, and PDX220 cells that were treated for 7 d with DMSO (Ctrl); 2 μg/ml Dox; 5–10 μM CQ and 2.5 μM palbociclib (P/CQ); or D/P/CQ. Control and Dox-treated sample percentages are continued from Fig. 3 B; experiments were performed at the same time as P/CQ- and D/P/CQ-treated samples (n = 3 from two independent experiments, mean ± SEM). *, P < 0.05; **, P < 0.01; ns, not significant by Student’s two-sided t test. (E) Immunoblot analysis of lysates generated from Tet-On c-MYC–expressing cells that were treated for 7 d with DMSO (Ctrl), 2 μg/ml Dox (D), 5–10 μM CQ, 2.5 μM palbociclib (P), or the indicated combinations (representative blots from two independent experiments). Lysates were blotted for c-MYC, pRB (phospho-S807/811), RB, CCNB1, CCNA2, and ACTB. Densitometry quantitation of c-MYC and pRB protein levels normalized to actin are shown. Source data are available for this figure: SourceData F5.