Fig. 2: Mitochondrial activity is critical for EC sprouting in 3D sprouting models.

HUVECs were transfected with control, Tfam, Cox10 or Trx2 siRNAs. 48 h after transfection, cells were subjected to various assays. a, b EdU incorporation assays for EC proliferation. ECs were stained with anti-VE-cad with counterstaining by DAPI while EdU was detected by a Click-iT assay. % EdU⁺ ECs were quantified (b). 10 random fields were counted for each group. Data are duplicates from and three independent experiments. c–e Cells were plated in fibronectin-coated Transwell filter and incubated for 4 h and 8 h. Migrated cells were visualized by crystal violet staining and quantified. 10 random fields were counted for each group. Data are duplicates from and three independent experiments. f–h GFP-expressing HUVECs were transfected with control, Tfam, Cox10 or Trx2 siRNAs. 48 h after transfection, cells were applied to spheroid sprouting assays in the absence or presence of mito-TEMPO (10 μM). Quantification of the sprout number per spheroid (g) and mean sprout lengths (h) on day 7. 10 spheroids per sample were counted. Data are duplicates from and three independent experiments. I, k GFP-expressing HUVEC spheroids were cultured in the presence of rotenone (2.5 μM), antimycin (2.5 μM), oligomycin (5 μM) or menadione (1 μM). Quantification of the sprout number per spheroid (j) and mean sprout lengths (k) on day 7. 10 spheroids per sample were counted. Data are duplicates from and three independent experiments. Data are duplicates from and three independent experiments. Data are means ± SEM. P values are indicated, using one-way ANOVA followed by Tukey’s multiple comparisons test (b, d, e, j, k) or two-way ANOVA followed by Sidak’s multiple comparisons test (g, h). Scale bar: 10 μm (a); 50 μm (c, f, i). Source data are provided as a Source Data file.