FIGURE 6.

AMPKα and VEGFR2 are required for endorepellin-mediated autophagy in HUVEC. A–D, immunofluorescence images depicting the co-localization of Beclin 1 (green) and LC3-II (red) shown upon treatment with vehicle (n = 31 cells from three biological replicates obtained from three independent experiments), 200 nm endorepellin for 6 h (n = 36 cells from three biological replicates obtained from three independent experiments), 30 μm compound C for 30-min pretreatment + 6 h (n = 28 cells from three biological replicates obtained from three independent experiments), or compound C + endorepellin (n = 28 cells from three biological replicates obtained from three independent experiments) in HUVEC. Nuclei are stained with DAPI (blue). Scale bar = 10 μm. White arrows indicate autophagosomes. E, quantification of the number of autophagosomes per cell in HUVEC treated according to the conditions shown in A–D. F, representative immunoblots of HUVEC pretreated with scrambled siRNA (SiScr) or siRNA targeting VEGFR2 (siVEGFR2) following treatment with endorepellin (200 nm, 6 h). G–H, quantification of VEGFR2 after normalization to GAPDH (one technical replicate from each of three biological replicates obtained from three independent experiments) and P-AMPKα:AMPKα ratio (one technical replicate from each of three biological replicates obtained from three independent experiments) shown in F. *, p < 0.05; **, p < 0.01; ***, p < 0.001; Student's t test.