Figure 3
ATR is required for MMc-induced COL6 secretion. (a) COL6 in the culture supernatants was detected by Western blotting. HRGECs were cultured with DMSO (control), ATRi or ATMi for 1 h, then MMc (30 µg/mL) was added in the cultures. Twenty-four h after MMc addition, the supernatants were collected. The lower panel showed CBB stain of the same membrane as a loading control. (b) HRGECs were cultured with DMSO (control) or ATRi for 1 h, then MMc (12 µg/mL) was added in the cultures. Two h after MMc addition, the cells were washed and cultured with or without ATRi for the indicated number of hours. The cells were then immunostained with anti-COL6 and anti- γ-H2AX antibodies. Scale bar: 50 µm in each panel. (c) The percentage of cells with cytoplasmic COL6 (COL6-positive cells) was measured using the samples of b by fluorescent images. (d) HRGECs treated with DMSO, CPT (1.0 µM, 4 h), NCS (50 ng/mL, 4 h) or MMc (12 µg/mL, 2 h) were washed, and cultured without DNA-damaging agents for the indicated number of hours. Cells were immunostained with anti-COL6 antibody. Scale bar: 50 µm in each panel. (e) The percentage of cells with cytoplasmic COL6 (COL6-positive cells) was measured using the samples of d by fluorescent images. A total of more than 100 cells of four independent fields per experiment was analyzed. (c, e) Statistical significance was determined by the Mann–Whitney U test. *p < 0.05, **p < 0.01.
