Fig. 7.

PFKFB3 overexpression restored impaired DNA repair in response to oxidative stress under high glucose by interaction with the MRN-ATM pathway. A and B Western blotting analysis of PFKFB3 and γH2AX. HUVECs were treated with Ads for PFKFB3 overexpression (ad-PFKFB3) or vehicle control (ad-ctrl) and then cultured in NG or HG for 48 h. C and D Western blotting analysis of ATM and MRN. After being infected with ad-PFKFB3 or ad-ctrl and then cultured in NG or HG for 48 h, HUVECs were subjected to H₂O₂ (300 μM for 30 min, 2 h recovery). E and F DNA damage levels assessed by comet assay. HUVECs were treated in the same conditions as (C) and (D). The representative images (E, scale bar: 100 μm) and the scatter dot plot of the tail moment per cell (F) are shown. G and H Cellular senescence detected by SA‐β‐Gal staining. After being treated with H₂O₂ (700 μM for 30 min), HUVECs were infected with Ads and then cultured in NG or HG for 48 h. Scale bar, 100 μm. I and J 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. K HUVECs were treated with ad-PFKFB3 or ad-ctrl for 48 h as indicated, and then subjected to H₂O₂ (300 μM for 30 min, 2 h recovery). Cell lysates (left, INPUT) and immunoprecipitated PFKFB3 (right, IP: PFKFB3) was immunoblotted with ATM and MRN antibody. L Confocal analysis of γH2AX, ATM and MRN foci. After being infected with ad-PFKFB3 or ad-ctrl and then cultured in HG for 48 h, HUVECs were subjected to H₂O₂ (300 μM for 30 min, 2 h recovery). 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.