Quercetin

The vascular rings were divided into 3 groups: control group, H₂O₂ group cultured in DMEM, and H₂O₂ + quercetin group cultured in DMEM containing 200 umol/L quercetin (Abcam, UK). After 1 hour, the H₂O₂ group and H₂O₂ + quercetin group were treated with 100 umol/L H₂O₂. After 4 hours, the culture supernatant was recovered, and then, the NO content was detected by the nitric oxide (NO) kit (Solarbio, China). Another 3 groups, treated as above, after 24 h, were treated by 100 umol/L H₂O₂. Then, NO, SOD, and MDA contents were detected using the relevant kits (Solarbio, China). In the mechanical damage experiment, shear the veins by scissors to form a 2 mm incised wound. The control group did not do any treatment. The wound group was cultured in DMEM, and the wound + quercetin group was cultured in DMEM containing 200 umol/L quercetin. After 24 hours, the veins were collected, and mRMA levels of IL-6, TNFα, CCL20, PCNA, and VEGF were detected by qRT-PCR. Then, the veins were collected for H&E staining, PCNA, and VEGF immunofluorescent staining.

Five compounds known to inhibit PI3K activity, including LY294002 (Sigma-Aldrich)^{48 (link), 49 (link)}, quercetin (Abcam)^{50 (link)}, wortmannin (Cell Signaling Technology)^{51 (link)}, GNE493 (SYNkinase)^{34 (link)}, and GDC-0941 (Abcam)^{38 (link)} were used for initial in vitro screening in a colony formation assay. GDC-0941 was selected for tumor suppression assay in an OX model with oral administration.

Cycloheximide (CHX), MG132 and quercetin were purchased from Sigma‐Aldrich (USA). quercetin was dissolved in DMSO and then diluted in DMEM in vitro. In in vivo experiments, the solvent for quercetin was 25% hydroxypropyl‐β‐cyclodextrin.
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¹⁷ (link) Anti‐RhoC and SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) were from Abcam (UK). Anti‐NEDD4 E3 ubiquitin protein ligase (NEDD4) and anti‐GAPDH antibodies were obtained from Cell Signaling Technology (USA). The anti‐WW domain containing E3 ubiquitin protein ligase 1 (WWP1) antibody was purchased from Proteintech (China).