M5293

Organoids were incubated with alpelisib, fulvestrant or a combination for 6 days and then washed with cold 1 × PBS. These cells were suspended in RIPA lysis solution (Biyuntian, P0013C) containing 1% protein and phosphorylation inhibitor (Abmole bioscience, M5293) on ice for 30 min. Then, the cells were centrifuged at 13,000 g at 4°C for 15 min and the supernatants were collected. The concentration was detected by the bicinchoninic acid (BCA) concentration detection kit (Biyuntian, P0012). The remaining parts were mixed in 5× loading buffer (Biyuntian, P0015), boiled for 10 min in a metal water bath, and then subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). After electrophoresis, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and incubated against primary antibody for 24 h. Membranes were washed with tris buffer saline‐Tween (TBS‐T) and incubated with a 1:5000 dilution of secondary antibody for 2 h. Protein bands were then visualized by Ultra Luminol/Enhancer Reagent (New Cell & Molecular, China). The following antibodies were used: PI3K‐110α (Abcam, ab1678, 1:500), p‐AKT (CST, 4060S, 1:1000), AKT (CST, 4691S, 1:1000), p‐mTOR (CST, 5536S, 1:1000), mTOR (CST, 2983S, 1:1000), p‐S6 (CST, 4857S, 1:1000), ER (CST, 13258S, 1:500) and GAPDH (Proteintech, 60,004‐1‐Ig, 1:10000).

NP cell line was treated with PDA NPs alone (1 µg mL⁻¹, dissolved in PBS) for 24 h or stimulated with TBHP (100 µm) for 12 h pretreated with or without PDA NPs (1 µg mL⁻¹) for 24 h at 37 °C with 5% CO₂. Then, total proteins were isolated from cells using RIPA lysis buffer supplemented with phosphatase and protease inhibitors (M5293, M7528; Abmole, China). Following quantification by BCA assay (Thermo Fisher Scientific, Inc.), being added with 5× protein loading buffer and being boiled at 60 °C for 10 min, equal quantities of extracted proteins (20–30 µg) were subjected to WB analysis. Primary antibodies against OXPHOS (cat. no. ab110413; mouse mAb) were bought from Abcam (Cambridge, UK) (n = 3 for each group).

The pCAGGS expression vector carrying the MARV GP gene and Strep purification tag was introduced into 293F cells. The cell density before transfection was 2 × 10⁶ cells/ml. The transfection dose of the expression vector was 1 mg/L. At7 days post-transfection, the 293F cells were resuspended in binding buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH = 8) and protease inhibitor cocktail (M5293, AbMole) was added. After sonication, 11 U/mg of avidin was added and incubated in an ice bath for 30 min. Purification was performed using StrepTrap XT affinity chromatography medium (Cytiva, 29.401.328 AC) on a protein chromatography system (GE, ÄKTA Pure). The expression and purification of MARV Angola GP1 was conducted analogously, but 10×His tag is used, and Ni²⁺ metal-chelating medium (SUNRESIN, C415320101) was used for affinity chromatography.