Fig. 7.

Senescence affects endogenous TR target AGO1x. (A) Images of senescence-associated β galactosidase assay and its quantification in MDA.MB.231 treated with 40 nM camptothecin (therapy-induced senescence (TIS)) or Vehicle (control) for 96 h. Data shows mean of three biological replicates and error bars show SD Students-t-test was performed and P value is indicated. (B) Relative mRNA levels of indicated mRNA in cells as in A. Data shows mean of three biological replicates and error bars show SD Students-t-test was performed and P value is indicated. (C) Immunofluorescence of AGO1x in MDA.MB.231 cells that were treated with 40 nM camptothecin (TIS) or Vehicle (control) or cells that were treated with camptothecin and left to recover for 14 days to allow bypass of senescence (Bypass of TIS). Scale bar=50 µm. A representative image of three biological replicates is shown. D. Quantification of results in (C). A minimum of 50 cells were scored for nuclear staining intensity using Image J. Statistical analysis was performed by ANOVA with Tukey post-test. A.U., arbitrary units. (E) Immunofluorescence of KI67 from MDA.MB.231 cells treated as in C. Scale bar=50 µm. A representative image of three biological replicates is shown. (F) Quantification of the percentage of cells expressing Ki67 shown in E. Statistical analysis was performed by ANOVA with Tukey post-test. (G) Immunoblot against the indicated proteins in MDA.MB.231 cells treated as in C. H3(pS10): phosphorylated Histone H3 at serine 10, and γH2AX: phosphorylated at serine 139 of histone H2AX variant. (H) Regulation of readthrough in senescent cells.