Ym3431

IHC staining and H-Score calculating were performed as our previous research [17 (link)]. The primary antibodies used in the assay were anti-RRAGB (1:300, 13,023–1-AP, Proteintech, USA), anti-p-mTOR (Ser2448) (1:200, YP0176, Immunoway, USA), anti-HIF1A (1:300, YT2133, Immunoway, USA), and anti-cleaved-caspase3 (1:100, YM3431, Immunoway, USA).

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.

SYBR Premix Ex Taq (RR820A) and Prime Script RT reagents (RR036A) were obtained from Takara Bio (Otsu, Japan). P-gp (mdr1), MRP1 (mpr1), BCRP (bcrp) and β-actin primers were additionally synthesized by Takara. Mouse anti-β-actin (3598.100, BioVision, Milpitas, CA, USA), rabbit anti-BCL-2 (12789-1-AP; Proteintec, Chicago, USA), rabbit anti-BAX (50599-2-lg; Proteintec), mouse anti-P53 (BM0101), rabbit anti-NF-κB (PB0161), rabbit anti-P-gp (BA1351‑2), anti-BCRP (BA2307‑2), anti-MRP1 (BA0567) (all from Boster, Wuhan, China), rabbit anti-cleaved caspase-3 (YM3431; ImmunoWay, Plano, TX, USA) and P-gp antibodies (MRK16, Mc-012; Kamiya Biomedical, Seattle WA, USA) were used. The chemotherapeutic drug ADM was purchased from Shenzhen Wanle Pharmaceutical Co. (Shenzhen, China) and ATO and Rsv acquired from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).