Fig. 3. S-2HG restrains the angiogenic activity of ECs.
a,b, S-2HG reduces the motile behaviour of cultured HUVECs in a scratch-wound assay. Quantification (a) and representative bright-field images (b) from control and S-2HG-treated HUVECs (n = 15 independent samples). c, Confocal images of phalloidin-labelled (grey) HUVEC spheroids showing reduced endothelial sprouting in S-2HG treated spheroids. Images were taken 24 h after treatment. d, PECAM1 (PECAM) immunofluorescence staining (grey) in P7 mouse retinas showing decreased vascular density after a single intravitreal injection of cell-permeable S-2HG at P5. Controls were obtained by injection of DMSO (vehicle, Ctrl) in the contralateral eye. A, artery; AF, angiogenic front; CP, capillary plexus; V, vein. e, Confocal images of P7 retinas from control and S-2HG-injected mice stained for ERG (cyan) and PECAM (red). f, Immunofluorescence staining for EdU (red), ERG (green) and PECAM (blue) in P7 mouse retinas of control and S-2HG mice. Proliferating (EdU and ERG double-positive) ECs are shown in yellow. g, Confocal images of retinas from control and S-2HG-injected mice stained for ICAM2 (ICAM, green), PECAM (blue) and collagen IV (COL, red). h, Immunofluorescence images of cleaved caspase-3 (yellow), ICAM (cyan) and PECAM (red) of P7 retinas from control and S-2HG-injected mice, excluding excessive apoptotic cell death in S-2HG-treated mice. Note that most of the cleaved caspase-3 signals come from nonvascular (ICAM/PECAM-negative) cells. i, Quantification of angiogenic parameters in retinas of control and S-2HG-injected P7 mice, as indicated (EC area: n = 21 samples each for control and S-2HG; EC proliferation: n = 24 each samples for control and S-2HG; regression index: n = 18 and 22 samples for control and S-2HG, respectively). For a and i, the data represent the mean ± s.e.m.; two-tailed unpaired t-test, ****P < 0.0001. The numerical data and P values are provided as source data.