Fig. 5
The PRC2 complex is critical for the deposition of heterochromatic histone marks and HP1 at telomeres. a Upon infection of U2OS cells with shRNAs against EZH2 and SUZ12, total protein was obtained and used for western blot detection of EZH2 and SUZ12. Actin was used as loading control. b U2OS were treated with increasing concentrations of the EZH2 inhibitor EPZ-6438 for 4 days. Nuclear protein extracts were used for western blot detection of H3K27me3. Actin was used as loading control. (Graph) Quantification is shown. c Representative images of the average number of colocalizations for TRF2 (green) and SUZ12 (red) in U2OS cells infected with a scramble or with EZH2 or SUZ12 shRNAs (left panel) or treated with vehicle or EZH2 inhibitor. Arrowheads indicate colocalization events. Scale bar, 10 μm. Below the images is shown the quantification of (left graphs) the total nuclear SUZ12 upon shRNAs or EZH2 inhibitor and (right graphs) the colocalization between TRF2 and SUZ12 (mean values ± s.e.m., n = number of cells). d Telomeric ChIP-dot-blot of the H3K27m3, H3K9m3, and H4K20m3 for U2OS cells infected with scramble, EZH2, or SUZ12 shRNAs. IgG was used as a control. IgG ChIP-dot-blot shared by different antibodies shows different exposure times according to the best exposure time required for each antibody. DNA input signal is also shown. Quantification of the immunoprecipitated telomeric repeat signal normalized by the input for each individual sample is shown below (mean values ± s.e.m., n = technical replicates). Student’s t-test was used for the statistical analysis (*p < 0.05, **p < 0.01, and ***p < 0.001)
