Fig. 4

DDR activation is linked to chromatin decondensation in the absence of LOXL2. a Dot graphs indicate the number of foci with γ-H2AX (left graph) and 53BP1 (right graph) per cell from control and LOXL2 KD cells treated with DMSO or the ATM inhibitor KU55933. b KAP-1 phosphorylation in control or LOXL2 KD cells was analyzed by western blot. Tubulin and total KAP-1 were used as loading controls. As a positive control, MDA-MB-231 cells treated with 0.1 µM doxorubicin for 8 h (to generate DSBs) were used. Intervening lanes were removed as indicated (left panel). Chk1 and Chk2 phosphorylation in control and LOXL2 KD cells were analyzed by western blot. Chk1 phosphorylation was detected using the anti-P(S317) Chk1 antibody. For phosphorylated Chk2, a shift was detected using an anti-total Chk2 antibody. GADPH was used as a loading control. All samples were obtained under the same experimental conditions; in addition, positive control samples (irradiated fibroblasts) were run on the same gel as their corresponding control and LOXL2 KD samples. Intervening lanes in the Chk1/2 and GADPH blots were removed as indicated (right panel). c Cell cycle profile of control and LOXL2 KD cells at 0, 2.5, 5, or 7.5 h after release from a double-thymidine block. Cells were analyzed by fluorescence activated cell sorting (FACS) after propidium iodide staining. d H3S10 phosphorylation levels in control and LOXL2 KD MDA-MB-231 cells were analyzed by western blot. Tubulin was used as a loading control. e Upper panel, MTT assay in control and LOXL2 KD MDA-MB-231 cells; lower panel, colony-survival assay in control and LOXL2 KD MDA-MB-231 cells. γ-H2AX staining and foci quantification are shown by immunofluorescence with the indicated antibody after H1 (f) or SUV-39H1 reinfection (g). MDA-MB-231 cells were infected with control or LOXL2 KD lentivirus, selected with puromycin, and reinfected with GFP (MOCK^GFP+), histone 1-GFP (H1^GFP+) (f) or SUV-39H1-GFP (SUV-39^GFP+) (g). Cells were fixed after 24 h. Dot graphs indicate the number of γ-H2AX foci per GFP-positive cell in each condition. Error bars indicate the SD from at least three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001