Hcclm3 lm3

Human L-02 hepatocytes and human HCC cell lines, including Hep3B, SK-Hep-1, and hepatoblastoma HepG2, were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). Human HCC cell line HCCLM3 (LM3) was purchased from ATCC (Manassas, VA, USA). Human HCC cell lines MHCC97H (97H) and Huh7 were purchased from BeNa Culture Collection (Beijing, China). HepG2, LM3, 97H, Huh7, and L-02 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Carlsbad, CA, USA), while Hep3B and SK cells were cultured in Eagle’s minimum essential medium (Gibco) replenished with 10% fetal bovine serum (FBS; HyClone Laboratories Inc., Novato, CA, USA) at 37°C with 5% CO₂. In addition, the HepG2, Hep3B, and LM3 cells were treated with human recombinant TGF-β1 (10 ng/ml, R&D Systems, Minneapolis, USA) for 48 h.

The HCC cell lines Hep3B, HCCLM3(LM3), HuH7, HB611, HepG2, SMMC7721, MHCC97-L (97-L), MHCC97-H (97-H), PLC/PRF/5 and BEL-7402 were purchased from the ATCC and maintained in Dulbecco’s modified Eagle’s medium or RPMI-1640 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere with 5% CO₂. The EGFR-WT and EGFR-I645L cDNAs were obtained from 97-L and 97-H cells following RNA isolation and subsequent reverse transcription PCR (Takara, Japan). cDNAs of wild type and mutanted EGFR were cloned into pCDH-EF1-coGFP-puro lentiviral vector (CD513B1, SBI Inc., Mountain View, CA, USA) using XbaI and NheI restriction sites, respectively. Lentivirus based shRNA against EGFR was purchased from Origene Inc. (TL320326, Rockville, MD, USA). For plasmid transfection, Lipofectamine 2000 was used according to the standard protocol. Plasmids were co-transfected with the packaging plasmid (TR30022, Origene, Rockville, MD, USA) into 293 T cells to generate the viral supernatant, then the viral supernatant infected cells. Stable HCC cell lines were established in culturing with 4 μg/ml puromycin (Thermo-Fisher, MA, USA).