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. 2021 May 7;9:656795. doi: 10.3389/fcell.2021.656795

FIGURE 6.

FIGURE 6

CDT causes nuclear distension associated with DNA damage in cycling cells of human colorectal organoids leading to decrease of growth. Human colorectal crypts were treated at day 0 with wild-type (2.5 and 25 ng/ml) or catalytic inactive mutant (H153A, 25 ng/ml, Mut) of CDT from E. coli for 16 h. (A) Representative images of organoids at day 8 with a bright-field microscope. Scale bar: 200 μm. (B) At days 4 and 8 of culture, organoid size was measured. N > 10 organoids. (C) EdU was added to the organoid culture medium for 16 h before fixation. Then, EdU was revealed and EdU-positive cells per organoid were quantified by confocal analysis. N > 6 organoids. (D) At days 4 (D4) and 8 (D8) of culture, organoid nucleus size was analyzed by confocal microscopy. N > 6 organoids. (E,F) At day 4 (D4) or 8 (D8), EdU was added to the organoid culture medium for 16 h before fixation and revealed. Then, γH2AX immunostaining was performed and γH2AX-positive cells in proliferating cell population (EdU+) from organoids were quantified. N > 6 organoids. (F) Representative images of EdU (green) and γH2AX (red) immunostaining in human colorectal organoids at days 4 (D4) and 8 (D8) treated or non-treated (NT) with 25 ng/ml of E. coli CDT were obtained from confocal analysis. Scale bar: 50 μm (mean ± SEM of at least three independent experiments) [∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ****P < 0.0001 versus non-treated (NT); ns, not significant].