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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 2. p130cas and VEGFR2 are internalized into autophagosomes and the nucleus, followed by caspase-10 cleavage, in ECs treated with Bev.

Figure 2.

(A) Representative confocal images showing expression of VEGFR2 (green) in Bev-sensitive RF24-par and Bev-resistant RF24-Bev cells treated with VEGF only or VEGF + Bev. Scale bar, 50 μm; n = 3.

(B) Expression of VEGFR2 pY1175 and pY1214 and total VEGFR2 in subcellular fractions of HPAECs. Only under VEGF-A (10 ng/mL) + Bev (5 μg/μL) treatment did the ~100-kD fragment of VEGFR2 appear together with the phosphorylated and total matured VEGFR2 (~220 kD). We used lamin A/C (LMNC) as a marker for the nuclear fraction (NER) and β-actin as a marker for whole-cell lysate (WCL) and cytoplasmic (Cyto) fractions.

(C and D) Expression of Cyto p130cas and its 31-kDa nuclear fragment was observed in subcellular fractions from RF24-par cells but not from RF24-Bev cells (C). We used lamin B1 (LMNB1) as a marker for NER and β-actin as a marker for Cyto. When treated with VEGF + Bev, the 100-kDa nuclear fragment of VEGFR2 was observed only in subcellular fractions of RF24-par cells but not of RF24-Bev cells (D). Activated/cleaved caspase-10 was also observed in the Cyto and NER fractions of RF24-par cells treated with VEGF + Bev.

(E and F) Representative confocal images showing co-localization of LC3B (green) and VEGFR2 (red) in RF24-par (E) or caspase-10-depleted RF24casp10—/— (F) cells in response to treatment with CTL, VEGF only, or VEGF + Bev. Scale bar, 50 μm; n = 3.

(G) Top: co-immunoprecipitation of p130cas and LC3B or VEGFR2 in RF24-par cells treated with CTL, VEGF, or VEGF + Bev. Bottom: reciprocal immunoprecipitates of VEGFR2 and LC3B or p130cas.

(H) Representative confocal images showing co-localization of LC3B and VEGFR2 or p130cas in RF24-par cells treated with CTL, VEGF only, or VEGF + Bev. Scale bar, 50 μm; n = 3.

(I) Representative transmission electron microscopy images of mouse ovarian ECs (MOECs). In comparison with VEGF treatment, in which nuclei (NUs), rough endoplasmic reticulum (RER), and regular mitochondria (Ms) were visible, VEGF + B20 (murine AVA) treatment induced numerous autophagosomes (APs), lysosomes (Ly), and autolysosomes (Aly); abundant phagophores (Ph) were identified in Aly membranes. The substructure was observed under 5,000× and 50,000× magnification (n = 5).