Fig. 4: ZNF768 is depleted upon senescence entry and its overexpression contributes to bypass this process.
A Protein lysates were prepared from IMR90 cells at Low or High population doublings. Western blot analyses were performed for the indicated proteins. Representative pictures of the cells are shown on the right part of the panel (Scale: 50 µm). B IMR90 cells were transduced with lentiviruses to overexpress a constitutively active form of RASG12V. Protein lysates were prepared at the indicated time following the overexpression and western blot analyses performed for the indicated proteins. Representative pictures of the cells are shown on the right part of the panel (Scale: 50 µm). C IMR90 cells were treated overnight with doxorubicin (0.1 µM). The cells were next washed and followed for the indicated times. Western blot analyses were performed for the indicated proteins. Representative pictures of the cells are shown on the right part of the panel (Scale: 50 µm). D Primary MEFs were transduced with retroviruses expressing different combinations of FLAG-ZNF768, RASG12V, or empty vector. After antibiotic selection, cells were maintained in culture until the first signs of senescence bypass were visible. Cells were then fixed and SA-β-gal assays were performed. The percentage of SA-β-gal positive cells is shown. The result presented is the average of four independent experiments (n = 4). Representative images are shown (Scale: 100 µm). E IMR90 cells were transduced with retroviruses expressing different combinations of FLAG-ZNF768, shGFP, shp53, RASG12V, or empty vector. After antibiotic selection, cells were maintained in culture for 8 days. Cells were then fixed and SA-β-gal assays were performed. The percentage of SA-β-gal positive cells from three independent counts are indicated and representative images are shown for each condition (Scale: 100 µm). In panels D and E, mean values were compared through a one-way ANOVA with Tukey’s multiple-comparisons test. Details about reproducibility are provided in the Statistics and reproducibility included in the Methods section. Source data are provided as a Source data file.
