Figure 4
From: PML is required for telomere stability in non-neoplastic human cells
PML associates with damaged telomeres. (a) Representative immuno-FISH experiments on WI38 normal human fibroblasts, before and after specific telomeric damage induced by RHPS4 treatment. Merged images generated by a telomeric PNA probe (red, Cy3) and anti-γH2AX (green, Alexa488) or anti-PML (blue, Cy5) antibodies. Enlargements of triple overlapping signals are shown on the right. (b) Quantitative analysis of the immune-FISH experiments shown in (a). Each dot represents one of fifty cells analyzed in each of three experiments and counted before (CTRL) and after RPHS4 treatment. The average values (red lines) show the number of TIFs per cell (1.77±0.36 before and 3.25±0.43 after treatment) or PML-NBs per cell (5.9±0.7 before and 9.78±1.39 after treatment), the percentage of PML-NBs colocalizing with telomeres (%PML/Tel: 8.38±2.25 before and 24.77±3.55 after treatment) and the percentage of PML-NBs colocalizing with TIFs (%PML/TIFs: 1.44±1.15 before and 8.88±2.37 after treatment) as detected by triple γH2AX/telomeres/PML signals colocalization. (c) Representative immuno-FISH experiment, performed as in (a), on WI38 cells before (CTRL) after (TRF2 KD) TRF2 RNA interference. Three enlargements of triple overlapping signals are shown. (d) Quantitative analysis, as in (b), of immune-FISH experiments performed on WI38 cells before (CTRL) after (TRF2 KD) TRF2 RNA interference. TIFs 0.72±0.13 in control and 9.04±0.81 in TRF2 KD cells; PML-NBs 14.0±1.14 in control and 17.0±1.25 in TRF2 KD cells; % PML/Tel 5.28±0.94 in control and 29.5±2.44 in TRF2 KD cells; % PML/TIFs 1.75±0.57 in control and 23.99±2.26 in TRF2 KD cells. The P-values indicated in (b) and (d) are based on a two-tailed Student’s t-test.
