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. 2021 Dec 2;10(12):bio058688. doi: 10.1242/bio.058688

Fig. 5.

Fig. 5.

RB pathway activation reduces readthrough. (A,B) Normalized Fluc/Rluc ratios of IMR-90 cells transduced with luciferase reporters UGA (A) or AQP4 (B) and treated with vehicle and/or 1 μM of palbociclib (Palbo) and/or 900 μg/ml of gentamicin sulfate (Gen) for 5 days before luciferase measurements. Error bars indicate SD of three independent experiments, n=3. (C) Normalized Fluc/Rluc ratios of IMR-90 fibroblasts transduced with an empty vector (Vector) or H-RasV12 oncogene (H-RasV12) to induce OIS but cultured for 35 days until they bypassed the phenotype (D35) and with AQP4 luciferase reporter. Cells were treated with vehicle (Ctrl) or 1 μM of palbociclib (Palbo) for 5 days before measuring luciferase activities at day 35 post-infection. Normalizations are presented as means relative to vector-infected cells, n=3 with technical triplicates for each experiment. One-way ANOVA with post-hoc Tukey HSD. Error bars indicate SD of biological triplicates. Tukey HSD P-values indicate that *=P<0.05, **=P<0.01 are significantly different. (D) Growth curves of proliferating (Vector) and ras bypassed (H-RasV12 D35) IMR-90 cells treated with vehicle (Control) or 1 μM of palbociclib (Palbo) for 5 days are shown. Data are presented as means normalized to day 0 of each condition and error bars indicate SD of three independent experiments, n=3. (E) Immunoblots for total RB (Tot RB) (note: upper band represents phosphorylated RB: pRB) and alpha-tubulin from non-senescent (V D35) and ras bypassed cells (R D35) following treatments with vehicle (Ctrl) or 1 μM of palbociclib (Palbo) for 5 days. Blots are representative of three independent experiments with similar results. (F) SA-β-gal of proliferating (Vector D35) IMR-90 cells and IMR-90 cells that have by-passed the senescent stage (H-RasV12 D35) treated for 5 days with vehicle (Ctrl) or 1 μM of palbociclib (Palbo) and fixed at day 35 (D35) post-infection. Data were quantified from many fields within one experiment to represent the entire petri dish. Three independent cell counts up to a total of at least 100 cells are presented as the mean and SD of positive cells. (G) Immunoblots for indicated proteins at day 7 post infection with an empty vector (Vect), pBABE-RPS14(WT)-Myc (RPS14) or pBABE-RPL22(WT)-Myc (RPL22): Myc (Myc-tag), total RB [RB (tot)], MCM6, phosphorylated H3 on serine 10 [H3(pS10)], total p53 [p53(tot)] and alpha-tubulin. Blots are representative of three independent experiments with similar results. (H,I) Normalized Fluc/Rluc ratios of IMR-90 cells transduced with an empty vector (Vector), or pBABE-RPS14(WT)-Myc (RPS14, H), or pBABE-RPL22(WT)-Myc (RPL22, I), and with the luciferase reporter AQP4. Luciferase activities were measured at days 7, 12 and 14 post infection. Normalizations are presented as means relative to vector-infected cells, n=3 with technical triplicates for each experiment Unpaired t-tests with equal s.d. were performed. Error bars indicate SD of biological triplicates. *=P<0.05, **=P<0.01, ***=P<0.001 are significantly different, using two-tailed Student's t-test. (J) Schema showing the RB activation/inhibition factors that modulate readthrough.