Yt3555

Cell immunofluorescence staining was conducted to evaluate the expression of proteins related to angiogenesis and cell apoptosis. HUVECs were plated in 6‐well plates (Corning, USA) at a density of 9.6 × 10⁵ cells and treated as mentioned above. Then, the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min, permeabilized and blocked with 0.5% Triton X‐100 (Biofroxx, Germany) and 1% BSA (Biosharp, Anhui, China) for 20 min. Then, the samples were incubated together with the following diluted vascular endothelial growth factor 2 (VEGFR2) (1:200, YT5845, Immunoway) and S6K (1:200, YT3555, Immunoway) primary antibody at 4°C overnight. Next, they were cultured with secondary antibody anti‐rabbit IgG [1:1000, 4413, Cell Signalling TECHNOLOGY(CST)] under ambient temperature for 2 h. Then, the cell nucleus was stained with Hoechst 33342 (Solarbio, Beijing, China), and the cytoskeleton was stained with phalloidin (Solarbio, Beijing, China). Samples were fixed with anti‐fluorescence quenching sealed tablets.

The total protein was separated by SDS-PAGE electrophoresis and transferred onto PVDF membrane (#IPVH00010, Millipore, USA). Then, the PVDF membrane was incubated with the following primary antibodies at 4 °C overnight: anti-RRAGB (1:1000, 13,023–1-AP, Proteintech, USA), anti-mTOR (1:500, 66,888–1-IG, Proteintech, USA), anti-p-mTOR (Ser2448) (1:1000, YP0176, Immunoway, USA), anti-S6K (1:1000, YT3555, Immunoway, USA), anti-p-S6K (Thr389) (1:1000, YP1427, Immunoway, USA), anti-HIF1A (1:1000, YT2133, Immunoway, USA), anti-pro-caspase3 (1:1000, AB32150, Abcam, USA), anti-cleaved-caspase3 (1:500, YM3431, Immunoway, USA), anti-PARP (1:1000, AB191217, Abcam, USA), anti-cleaved-PARP (1:1000, AB32064, Abcam, USA), and anti-β-tubulin (1:2000, 10,094–1-AP, Proteintech, USA). Subsequently, the PVDF membrane was incubated with the corresponding HRP labeled goat anti-mouse (1:500, BS12478, Bioworld, USA) or goat anti-rabbit IgG (1:500, BS13278, Bioworld, USA) at room temperature for 1 h. FDBio-Dura ECL Kit (FD8020, FDbio, China) was used to visualize the protein bands.