A-Type Lamins Maintain the Positional Stability of DNA Damage Repair Foci in Mammalian Nuclei
Figure 3
Altered lamin A/C-H2AX interaction after DNA damage.
(A) Example immunofluorescent images of lamin-Histone H2AX interactions measured by proximity ligation assay in murine embryonic fibroblasts. Red dots represent the PLA signal, produced from antibodies in close proximity (<40 nm), and blue shows DNA stained by Hoechst 33342 dye. (i) Negative controls containing an antibody against only one interaction partner (either lamin A/C, H2AX or γH2AX). (ii) Sample containing both lamin A/C and H2AX antibodies (iii) Sample containing both lamin A/C and γH2AX antibodies. Scale bars 20 µm. (B) Automated microscopy was used to automatically quantitate PLA signal −/+ DNA damage in LMNA−/− GFP-lamin A cells stained with Hoechst 33342. The images show one example field of cells each for − and + DNA damage, in two different fluorescent channels containing Hoechst 33342 staining (top) and PLA signal (middle). Images are overlayed with automated identification of: cells as objects (blue), cell nuclei (green) and PLA signal (red dots). The bottom panel shows a colour overlay of PLA (green dots) and Hoescht staining (blue). (C) The histograms show the number of PLA dots +/− s.e.m in >2000 cells from two independent experiments. (i–iii) show results from murine embryonic fibroblasts and (iv) is from human cells. (D) The image shows the location of the PLA signal obtained from interaction between lamin A/C and γH2AX relative to chromocenters demarked by Hoechst 33342 staining in one single nucleus. (E) GFP-lamin A - H2AX complexes were found by co-immuno-pulldown. LMNA−/− GFP-lamin A cells were lysed in NP40 buffer and GFP-lamin A was isolated using protein A/G beads and anti-GFP antibody. The eluted complexes were probed by western blot with antibodies against H2AX, IgG and lamin A/C. (F) GFP-lamin A - γH2AX complexes were found by co-immuno-pulldown. 293T cells, transfected with either GFP-lamin A or GFP alone, were processed as described in (E) and the eluted samples were probed by western blot with antibodies specific to GFP and γH2AX.