Bca protein concentration assay kit

The proteins of cells or tissue were extracted with RIPA lysis buffer (BIYUNTIAN, China). The protein concentration was measured using a BCA protein concentration assay kit (BIYUNTIAN, China) according to the instructions. Protein electrophoresis was carried out in 5–12% gradient gels. Then, the proteins were transferred onto a PVDF membrane. After membrane transfer, antibodies against FOXS1 (1:1000, Thermo, USA), E-cadherin (1:1000, Abcam, China) and N-cadherin (1:1000, Abcam, China) were incubated overnight at 4 °C. The HRP-labelled secondary antibody (1:50000, BOSTER, China) was then incubated for 2 h at room temperature. Protein bands were detected with an X-ray film and analysed.

Quercitrin (standard product, HPLC purity ≥ 98%, product number: SQ8050, penicillin mixture (100×)) was purchased from Solarbio (Beijing, China). DMSO was purchased from Solarbio (Beijing, China). DMEM and fetal bovine serum were purchased from Gibco (Grand Island, NY, USA). fetal bovine serum (FBS) and trypsin EDTA solution were purchased from BI (Shanghai, China). RNAiso Plus was purchased from TaKaRa (Dalian, China); fluorescent quantitative from Genstar (Beijing, China). CCK8 and HiScript III RT SuperMix (R312-01) were purchased from Novozymes (Nanjing, China); the ROS kit and BCA protein concentration assay kit were purchased from Biyuntian Biotechnology (Shanghai, China). Acetylcysteine (NAC) was purchased from (Shanghai, China). The Ultrasensitive ECL chemiluminescence kit (P10300) was purchased from Xin Saimei Biotechnology (Suzhou, China). AcH3 (#9649), AcH4 (#8647), NF-kB p65 (#6956), Phospho-NF-kB p65 (#8214S), p38 MAPK (#8690), Phospho-p38 MAPK (#8203S), IκB-α (#8219S), Phospho-IκB-α (#8219S), AKT (#4691T), Phospho-AKT (#9271T), Anti-mouse IgG (#7076P2), Anti-rabbit IgG (#7074S), β-actin (#4970) and HRP secondary antibodies (#7076, 7074) were purchased from CST (Mass, USA). IL-8 was purchased from Proteintech (Wuhan, China). iNOS was purchased from NOVUS (MN, USA). HO-1 (ab189491) and NQO1 (ab80588) were purchased from Abcam (Cambridge, UK).

MDA-MB-231 cells were gathered and the total proteins in each sample was obtained and quantified using the bicinchoninic acid (BCA) protein concentration assay kit (Biyuntian, Beijing, China) after treatment with different concentrations of CLE-10 (0, 5, 10, 15 µM) for 24 h. Equal amounts of protein (50 µg) was separated by 6–15% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Beijing Labgic Technology Co., Ltd.). Membranes were blocked with 5% milk (BD) for 2 h. After washing, the membranes were probed overnight using the specific primary antibodies LC3, PI3K (p85), AKT, mTOR, p-PI3K (Tyr 508), p-AKT (Ser473), p-mTOR, p-ULK1(Ser 757), p62, Bax, Bad, Bcl-2, and Bcl-xl (Cell Signaling Technology) at 4 °C, followed by secondary antibody. Chemiluminescence was performed with a chemiluminescence developing solution (Biyuntian, Beijing, China) on Kodak X-ray films or using a chemiluminescence image analysis system (Tanon 5200). LC3, p-ULK1, ULk1, p62, mTOR, and p-mTOR were also detected with or without an autophagy inhibitor (CQ, 3-MA) or the mTOR agonist rapamycin. Cells were treated with 3-MA (5 mM), CQ (40 µM), or rapamycin (100 nm), CLE-10 (15 µM), 3-MA (5 mM) + CLE-10 (10 µM), CQ (40 µM) + CLE-10 (15 µM), and rapamycin (100 nm) + CLE-10 (15 µM); each for 24 h. Western blot analysis was used to detect the relative protein expression as described above.