Anti actin

Total protein was extracted from cells using a Protein Extraction Kit (KaiJi, Tianjin, China) according to the manufacturer's instructions. The concentration of protein was quantified via bicinchoninic acid (BCA) protein assay (Beyotime, Shanghai, China) according to the manufacturer's instructions. Equal amounts of total protein from each sample (50 μg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membranes were incubated with rabbit anti-E-cadherin (1:1,000; Cell Signaling Technology, Boston, MA, USA), rabbit anti-HBc (1:1,000; Dako, Glostrup, Denmark), rabbit anti-NTCP (1:1,000; Sigma-Aldrich), rabbit anti-Na⁺-K⁺-ATPase (1:1,000; Abgent, San Diego, CA, USA), and anti-actin (1:1;000; Boster, Wuhan, China) primary antibodies at 4°C overnight, and subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000; Boster) at 37°C for 1 h. Blots were developed using an enhanced chemiluminescence reagent (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. The gray value of blots was calculated by the Image Lab.

Proteins from lysates were prepared and resolved by 12% SDS-PAGE at 100 V, transferred for 1.5 h at 250 mA onto Nitrocellulose membranes, and analysed by immunoblotting as described previously^{56 (link)}. The information for primary antibodies is as follows: anti-ferritin (cat# 69090), SdhA (cat# 137040), and SdhB (cat# 178423) from Abcam (Cambridge, MA, USA), anti-Xod (cat# 55156-1-AP), citrate synthase (cat# 16131-1-AP), aconitase 2 (Aco2) (cat# 11134-1-AP), Ndufs1 (cat# 12444-1-AP), Ndufs3 (cat# 15066-1-AP), Uqcrc1 (cat# 21705-1-AP), and Uqcrfs1 (cat# 1843-1-AP) from Proteintech Group Inc. (Chicago, IN, USA), anti-Actin (cat# BM0627) from Boster (Wuhan, China), anti-Tubulin (cat# T0198) from Sigma-Aldrich (St. Louis, MO, USA), anti-TfR1 antibody (cat# 136800) from Zymed (San Francisco, CA, USA), anti-IscU, Fech, Irp1, and Irp2, anti-Fxn (self-made)^{31 (link)}, anti-CytC (cat# 1896-1) from Epitmics (Burlingame, CA, USA), anti-Vdac (cat# 4661S) from Cell Signaling Technology Inc (Shanghai, China).

Ten thousand B16 cells per well were seeded into 6-well plates. Twenty-four hours later, the medium was refreshed and supplemented with dioscin (0, 0.1, 0.5, 1, 2 or 4 μM). After continuous treatment with dioscin for 48 h, drug-treated and untreated cells were washed twice with PBS and lysed in RIPA buffer (0.25 m Tris-HCl pH 6.8, 8% SDS, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin, 1.0 mg/mL leupeptin). The cell extracts were separated by 10% SDS-PAGE and immunoblotted using anti-Cx26 (#T0374; Epitomics), anti-Cx43 (#A0084; Abclonal) or anti-actin (#BM0626; Boster) antibody.