Fig. 3

Hyperlactylation of microglia promotes angiogenesis in vitro. a Lactate production of HMC3 cells in the three groups treated with hypoxia for 0, 12, and 24 h (n = 3 independent experiments, at least 6 mice each group). b Quantification of Pan-Kla in the HMC3 cells of hypoxia 0 h, hypoxia 12 h, and hypoxia 24 h were analyzed by Western blotting (n = 3 per group). c Tube formation analysis. HMC3 cells were pretreated with 1% O₂ or normoxia for 24 h and then cocultured with HRMECs; tube formation was assayed 12 h and 20 h after cell seeding (n = 3 independent experiments, 3 images for each group); scale bar, 50 µm. d HRMECs angiogenic capacity was evaluated by spheroid-sprouting assay. Representative images of spheroid sprouting were analyzed (n = 3 independent experiments, 3 images for each group); scale bar, 50 µm. e HRMECs migration was evaluated by Transwell assay (n = 3 independent experiments, 3 images for each group); scale bar, 25 µm. f HRMEC proliferation was evaluated by Ki67 staining (n = 3 independent experiments, 3 images for each group); scale bar, 100 µm. g, h Quantification of lactate and Pan-Kla in HMC3 cells in the three groups treated with DCA (20 mM), DMSO, and rotenone (50 nM) under hypoxia for 24 h (n = 3 per group). i–l HMC3 cells were pretreated with 1% O₂ for 24 h with DCA, DMSO, or rotenone. Then HRMECs were cocultured with the pretreated microglia. The tube formation, spheroid sprouting, migration, and proliferation assays were performed as shown in the “Methods” (n = 3 independent experiments, 3 images for each group); scale bars, 100 μm (k) or 50 μm (i, j, and l). *p < 0.05; **p < 0.01; ***p < 0.001