Epirubicin epi

Human immortal hepatic HL-7702 cells and HCC cell lines MHCCLM3 and SMMC-7721were obtained from the cell bank of Shanghai Institutes for Biological Sciences (Shanghai, China). Both cell lines were cultured in Dulbecco’s modified eagle medium (DMEM, GE Healthcare Life Sciences, Chicago, IL, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 100 U/ml penicillin and 100 μg/mL streptomycin, and grown in a 95% air and 5% CO₂ humidified atmosphere at 37°C. Camptothecin (CPT) (TCI chemicals, Shanghai, China), sorafenib (Selleck, TX, USA), doxorubicin (DOX), epirubicin (Epi) and verapamil (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in DMSO as stocking solution respectively, then diluted in sterile saline before use.

Lactic acid (LA), glycolic acid (GA), stannous octoate, hydroxyl-terminated PEG, combretastatin A4 disodium phosphate (CA4P) and epirubicin (EPI) were purchased from Sigma–Aldrich (USA). Primary antibodies, including anti-PCNA, anti-CD34, anti-CD31, anti-MTA1, anti-TGF-β, anti-CRT and anti-HMGB1, were obtained from Abcam (Cambridge, UK). Antibodies for flow cytometric analysis, including anti-CD45, anti-CD11c, anti-CD86, anti-CD11b, anti-Gr-1, anti-CD3, anti-CD4, anti-CD8 and anti-FOXP3 were purchased from Biolegend. The 4′,6-diamidino-2-phenylindole (DAPI) and TUNEL assay kit were purchased from Thermo Fisher Scientific, Waltham, MA, USA.

Epirubicin (EPI) and Cisplatin (CS) were purchased from Sigma-Aldrich and dissolved in water and DMSO, respectively. Phosphoramide mustard (PM), the active metabolite of Cyclophosphamide, from National Cancer Institute and dissolved in DMSO. Human recombinant LH was a gift from Merck KGaA (Germany).
The following primary antibodies were used: Tubulin (T9026, Sigma-Aldrich); GADPH (G9545, Sigma-Aldrich); LHCGR (sc-25828, Santa Cruz); FOXL2 (ab5096, Abcam); γH2AX (05-636, Millipore); pan-cytokeratin (pan-CK, Z0622, Dako); αSMA (A2547, Sigma-Aldrich); RAD51 (sc-8349, Santa Cruz Biotechnology); ERCC1 (sc-17809, Santa Cruz Biotechnology); MLH1 (sc-271978, Santa Cruz Biotechnology). HRP-conjugated secondary antibodies were as follows: donkey anti-goat (Jackson ImmunoResearch) (1:50000); goat anti-mouse (GE Healthcare) (1:5000); goat anti-rabbit (GE Healthcare) (1:5000). The following secondary antibodies were used for IF analysis: Cy3-labeled donkey anti-goat antibody (1:400); Alexa Fluor 488-labeled goat anti-mouse (Thermo Fisher Scientific) (1:500); Alexa Fluor 568-labeled goat anti-mouse (Thermo Fisher Scientific) (1:500); Alexa Fluor 488-labeled goat anti-rabbit (Thermo Fisher Scientific) (1:500).