Mcl 1

HepG2 cells were lysed in RIPA buffer (containing 1 mmol·L⁻¹ PMSF) (Beyotime, Shanghai, China). The protein samples were quantified using an enhanced BCA assay kit (Beyotime, Shanghai, China). Proteins were separated on a 10% or 15% SDS-PAGE gel by electrophoresis and then transferred to a 0.45 or 0.22 μm PVDF membrane (Millipore, Darmstadt, Germany). The membrane was blocked with 5% skim milk and incubated with primary antibodies for caspase-8, caspase-9, caspase-3, Bax, Bcl-2, Mcl-1, Bcl-XL, survivin, MMP-7, MMP-9, and β-Actin (ABclonal, Wuhan, China) overnight at 4 °C and washed with TBST buffer three times. We then incubated the samples with HRP-conjugated goat-anti-rabbit IgG secondary antibody (ABclonal, Wuhan, China) and then washed with TBST buffer three times. Finally, we applied 1 mL of ECL agent (Advansta, Menlo Park, CA, USA) on the membrane, and the protein blots were developed using an Omega Lum G imaging system (Aplegen, Pleasanton, CA, USA). Image J software was used to scan the gray values of the blots.

MTT, Trizma^® base, glycine, SDS, acrylamide, N, N-Methylene bisacrylamide, and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM), phosphate buffer saline (PBS), and penicillin-streptomycin solution were from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). Doxorubicin hydrochloride was purchased from Aladdin (Shanghai, China). Rabbit polyclonal antibodies against STAT3, phospho-STAT3 (Tyr705), VEGF, MMP-7, Cyclin D1, Mcl-1, Survivin, Bcl-XL, Bcl-2, β-actin, and HRP-conjugated goat anti-rabbit antibody were purchased from ABclonal (Woburn, MA, USA). WesternBright™ Sirius chemiluminescent detection reagent was from Advansta (Menlo Park, CA, USA). Stock solutions of compounds (50 mM) were prepared in DMSO and stored at 4 °C. The stock solutions were diluted with DMEM to working concentrations as working solutions.