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. Author manuscript; available in PMC: 2022 Feb 21.
Published in final edited form as: Cell Rep. 2022 Jan 25;38(4):110301. doi: 10.1016/j.celrep.2022.110301

Figure 3. Autophagy is essential for VEGFR2 internalization under AVA therapy.

Figure 3.

(A) Bevacizumab (Bev) induced enrichment of LC3B loci only in AVA-sensitive RF24-par cells, not in resistant RF24-Bev cells. Shown are representative confocal microscopy images for GFP-LC3B (green) expression in RF24-par or RF24-Bev cells in response to treatment with CTL, VEGF only, or VEGF + Bev. Scale bar, 50 μm; n = 3. Red arrows show the LC3B loci formed in the cells.

(B) In vitro transfection with the pMAP-LC3B CRISPR-Cas9 construct used to knock out LC3B and insertion of pMAP LC3B homology-directed DNA repair (HDR) in RF24-par endothelial cells (ECs).

(C) Expression or absence of LC3B in 3 CRISPR-Cas9 knockout (KO) cells. β-Actin was used as a loading CTL.

(D) Representative confocal microscopy images of VEGFR2 (green) in RF24-par WT cells or LC3B2 CRISPR-Cas9 KO cells treated with VEGF + Bev. Scale bar, 50 μm; n = 3.

(E–G) Autophagy inhibition by hydroxychloroquine (HCQ) reduced VEGFR2 internalization into the LC3B lysosomal compartment and NU in RF24 cells. RF24-par cells pre-treated with 40μM HCQ (24 h) were subjected to VEGF-A alone or Bev + VEGF-A treatment for another 48 h.

(E) LC3B loci in cells were first determined by immunofluorescence staining with an anti-LC3B antibody. Scale bar, 50μm.

(F) The autophagy flux in RF24-par cells with or without HCQ treatment was measured by acridine orange (AO) staining and analyzed with fluorescence-activated cell sorting (FACS). The percentage of acidic vesicle organelles (AVOs) was statistically compared between CTL and HCQ or VEGF-A + Bev and HCQ + VEGF-A + Bev; two-tailed Student’s t test. Data are expressed as mean ± SD (n = 3).

(G) Representative images of confocal microscopy on RF24-par cells treated with HCQ or left untreated and stained with Hoechst (blue)/VE-cadherin(green)/VEGFR2 (red) for each group (CTL, VEGF, and VEGF + Bev). Each experiment was repeated at least three times, and representative images from 15 high-power fields in each condition are shown. Membrane VEGFR2 and VE-cadherin were present in RF24-par cells pretreated with HCQ + VEGF-A + Bev, where the nuclear VEGFR2 was minimal in comparison with the internalized VEGFR2 in Figure 2F and/or 2H. Scale bar, 50 μm; n = 3.