Fig. 2.

Clioquinol selectively down-regulates VEGFR2 in ECs. a Western blots showing VEGFR2, VEGFR1, Tie2, FGFR1, and β-actin expression in HUVECs after 4-hour treatment with 0, 2.5, 5, or 10 µM clioquinol. b-e Expression level (% of 0 µM) of VEGFR2 (b), VEGFR1 (c), Tie2 (d), and FGFR1 (e) normalized to β-actin in HUVECs treated as described in (a) (n = 3 independent experiments). f Mean fluorescence intensity (MFI) of membrane VEGFR2 on HUVECs treated with or without 10 µM clioquinol for 0.5, 1, 2, 3, and 4 h, as assessed by flow cytometry (n = 4). g Phase-contrast microscopic images of HUVEC spheroids after 24-hour treatment without or with clioquinol in the absence or presence of 25 ng/mL VEGF. Scale bar: 135 μm. h Sprouting (% of control) of HUVEC spheroids treated as described in (g) (n = 13–15). i Western blots showing VEGFR2 and β-actin expression in HUVECs, HDMECs, hPC-PLs, NHDFs, MCF-7, MDA-MB-231, and 4T1-Luc2 cells. j Expression level (% of HUVEC) of VEGFR2 normalized to β-actin in different cell types as described in (i) (n = 3 independent experiments). k Correlation between cell viability and VEGFR2 expression following exposure to 10 or 25 µM clioquinol. Means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. (b-e, h, j: one-way ANOVA with Tukey’s multiple comparisons test; f: unpaired Student’s t-test; k: Pearson correlation coefficient)