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

Fig. 1.

Fig. 1.

Readthrough is reduced in OIS. (A) Rluc (Renilla luciferase) in black is linked to Fluc (Firefly luciferase) in gray, by an intercistronic region in stripes. Rluc is the internal control of the gene, while Fluc is the readthrough sensor. A UGA codon within an artificial context or the AQP4 stop codon region is inserted in the intercistronic region (left). The expression of Fluc indicates the efficiency of readthrough. The intercistronic region from the non-readthrough control lacks the stop codon (TGA mutated by CGA) (right). (B) Immunoblots for Rluc in IMR-90 cells transduced with UGA and AQP4 reporters (Rt) or with the non-readthrough controls (No Rt), 12 days post infection. Alpha-Tubulin was used as a loading control. *, non-specific bands at 48 and 90 kDa; n=3. (C) Polysome profiles were performed 12 days post infection with an empty vector (Vector) or H-Ras V12 oncogene (H-RasV12) showing that global translation is similar in senescent and non-senescent IMR-90 cells, n=3. (D) Rluc plots indicate cap-dependent translation in IMR-90 cells with an empty vector (Vector) or with the oncogene H-Ras V12 (H-RasV12) at day 5 (D5, still proliferating), 12 (D12, senescent cells) or 20 (D20, senescent cells). Error bars indicate s.d of three independent experiments, n=3. (E) Fluc plots indicate the decrease of readthrough-dependent-translation of cells as in D. Error bars indicate s.d. of three independent experiments, n=3. (F) The percent of readthrough was calculated in cells as in D by dividing the Fluc/Rluc ratio from UGA or AQP4 luciferase reporters by the Fluc/Rluc ratio from non-readthrough controls multiplied by 100, n=3. (G) Fluc/Rluc ratios with data as in F but normalized relative to empty vector-infected cells n=3. (H) Immunoblots of H-Ras, total RB (Tot RB), total p53 (Tot p53), MCM6, phosphorylated Histone H3 on serine 10 [H3(pS10)] and tubulin at days 5, 12 and 20 post infection with an empty vector (V) or H-RasV12 oncogene (R), n=3, representative blot is shown. For panels E, F and G, significance was tested with one-way ANOVA with post-hoc Tukey HSD tests. Error bars indicate SD of three independent experiments. Tukey HSD P-values indicate that *=P<0.05 and **P<0.01.