Figure 5
From: PML is required for telomere stability in non-neoplastic human cells
PML depletion leads to telomere damage and senescence in human fibroblasts. (a) Growth of WI38 and MRC5 cells infected with PML shRNAs KD1 or KD2 or control luciferase shRNA (CTRL). Cells were counted at the indicated times after 6 days of puromycin selection. The error bars indicate the s.d. of the counts obtained in three experiments. Above the curves is a representative western blotting with the indicated antibodies, from cells lysed at day 3 after selection. (b) Microphotographs of control (CTRL) or PML knockdown (PML KD1) WI38 cells at 6 days post selection, unstained or stained for SA-β-galactosidase. (c) Quantitative analysis of SA-β-gal-staining on the cells shown in (b). (d) Western blotting with the indicated antibodies on the indicated WI38 cells lysed after 6 days of PML RNAi. (e) Representative immuno-FISH experiment on MRC-5 cells infected with PML-KD1: merged image of the signals obtained with a telomeric probe (red, Cy3) and anti-yH2AX (green, Alexa488) and anti-PML (blue, Cy5) antibodies. Enlarged PML/TIF colocalization images are shown on the right. (f) Quantitative analysis of immuno-FISH experiments performed as in (e), showing the number of TIFs per cell in MRC5 and WI38 cells, before (CTRL) and after PML knockdown (PML KD). Each dot represents one of the fifty cells analyzed in each of three experiments. The average value (red line) shows the number of TIFs per cell (0.96±0.19 in Control and 2.9±0.36 PML KD MRC5 cells and 1.34±0.19 in control or 2.6±0.3 in PML KD WI38 cells, respectively). The P-value was obtained by a two-tailed Student’s t-test. (g) Representative immuno-FISH experiment performed on PML-depleted MRC-5 cells using a telomeric probe (red, Cy3) and anti-p53 (green, Alexa488) and anti-γH2AX (blue, Cy5) antibodies; notably, very few apoptotic nuclei were detected in PML-depleted cells without a significant increase as compared with control cells. Enlargements of a single p53-positive cell and TIFs are shown on the right. (h) Quantitative evaluation of p53-positive MRC5 cells after the infection with a control vector (CTRL) or the PML-KD1 vector. (i) Quantitative analysis of immuno-FISH experiments indicating the percentage of p53-positive cells showing the indicated number of TIFs per cell. The experiments shown in (h) and (i) are representative of three.
