Fig. 6.

FOXO1 knockdown improves DNA repair mediated by the MRN-ATM pathway via PFKFB3 activation. A and B Western blot analysis of PFKFB3. After being infected with sh-FOXO1 or sh-ctrl and then cultured in NG or HG for 48 h, HUVECs were subjected to H₂O₂ (300 μM for 30 min) and harvested at the indicated time points. C Confocal analysis of the recruitment of PFKFB3 to DNA damage sites γH2AX foci and the colocalization between PFKFB3 and ATM in HUVECs upon H₂O₂ (300 μM for 30 min, 2 h recovery). White arrows indicate co-localized foci. Scale bar, 10 μm. D and E DNA damage levels assessed by comet assay. After being infected with Ads for PFKFB3 knockdown (sh-PFKFB3), sh-FOXO1 or sh-ctrl and then cultured in NG or HG for 48 h, HUVECs were subjected to H₂O₂ (300 μM for 30 min, 2 h recovery). The representative images (D, scale bar: 100 μm) and the scatter dot plot of the tail moment per cell (E) are shown. F and G Cellular senescence in HUVECs. After being treated with H₂O₂ (700 μM for 30 min), HUVECs were infected with the indicated Ads and then cultured in NG or HG for 48 h. Scale bar, 100 μm. H and I HR and NHEJ activity. After being treated with DNA repair reporter (DRR) assay, HUVECs were infected with Ads and then cultured in NG or HG for 48 h. J and K Western blotting analysis of ATM, MRN and PFKFB3. HUVECs were treated in the same conditions as (D) and (E). L Confocal analysis of γH2AX, ATM and MRN foci. After being infected with sh-PFKFB3 or sh-ctrl for 48 h, HUVECs were subjected to H₂O₂ (300 μM for 30 min, 2 h recovery), or left untreated. Scale bar, 10 μm. Each experiment was repeated independently at least three times. All results are displayed as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA analysis.