Gefitinib

Compound 968 was purchased from Calbiochem (Merck Millipore, Darmstadt, Germany). erlotinib hydrochloride was obtained from Biovision Inc. Compound 968, erlotinib, gefitinib, and cisplatin were dissolved in DMSO, and used at the indicated concentrations. Antibodies against β-actin and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal EGFR antibody was obtained from Becton, Dickinson and Company. Rabbit polyclonal GAC antibody was made in a company. RPMI 1640 Medium (with 300 mg/L L-Glutamine) and Advanced RPMI 1640 Medium (without L-Glutamine) were purchased from GIBICO (Carlsbad, CA, USA). Crystal violet and other analytical grade chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Antibodies against AnxA6, as well as secondary anti-mouse, anti-goat, and anti-rabbit horseradish peroxidase-conjugated antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies against COXIV, Cytc. C, Pyruvate dehydrogenase (PDH), and Superoxide dismustase (SOD1) were purchased from Cell Signaling Technology (Beverly, MA, USA). Oil Red-O, Rotenone, 2-Deoxy-D-Glucose, Etomoxir, and antibody against β-actin (ACTB) were purchased from Sigma Aldrich (St. Louis, MO, USA). A set of EGFR-TKIs including lapatinib-ditosylate, erlotinib, gefitinib, and canertinib were purchased from BioVision (Milpitas, CA, USA). The Mito Stress Test Assay, Palmitate FAO Assay, and the culture media were purchased from Agilent Technologies (Santa Clara, CA, USA). Except where otherwise indicated, all the other reagents were purchased from Sigma Aldrich and/or Cell Signaling Technology.

Trametinib, ICG-001, PRI-724, dabrafenib, GDC-0994, PD318088, selumetinib were purchased from Selleckchem. Erlotinib, SCH772984 and RAF265 were purchased from Cayman Chemical. Gefitinib was purchased from BioVision. XAV939 and CHIR99021 were purchased from Merck Millipore.

Temozolomide (TMZ, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) to prepare a stock solution of 100 mM. Human ANGPTL4 protein (Fc Tag) (Sino Biological, Wayne, PA, USA) was dissolved in sterile water to prepare a stock solution of 250 μg/mL. Gefitinib (Abcam, Cambridge, UK), AKT (Abcam), PI3K (Abcam), and ERK inhibitors (Abcam) were dissolved in DMSO.

Cells were grown in 35 mm dishes. MDA-MB-468 cells were treated with different doses of EGF for different time points as mentioned in the results section. Whenever required, cells were treated with pathway inhibitor Gefitinib (Abcam ab142052, Cambridge, UK). After treatment, cells were lysed in RIPA buffer containing PMSF (1 mM), sodium orthovanadate (1 mM), sodium fluoride (50 mM) and EDTA (1 mM). Total protein was estimated by Lowry’s method [40 (link)]. An equal amount of lysate from each sample was resolved by SDS PAGE and transferred to PVDF membrane by wet transfer. The membrane was blocked by 3% BSA in TBST for 2 h, followed by overnight incubation with primary antibody at 4 °C. Target proteins were detected by chemiluminescence (SuperSignal West Dura kit, Thermo Fisher Scientific, Waltham, MA, USA) using HRP conjugated secondary antibody. Developed blots were imaged using a gel documentation system (ChemiDoc XRS+, BioRad, Hercules, CA, USA). Detected bands were quantified by densitometry using ImageJ [41 (link)]. Target proteins were normalized with respect to loading control. Details on the antibodies used are provided in Supplementary Table S3a.

To study the antiviral effect or cell viability of EGFR inhibitors, HepG2.2.15 cells near confluent growth (>90%) were treated with 0–12.5 μM erlotinib (ab141930, Abcam, Cambridge, UK) or 10 and 20 μM gefitinib (ab142052, Abcam) for 48 h. The media were replaced every 24 h. To study the influence of EGFR inhibitors on STAT3 phosphorylation, HepG2.2.15 cells were treated with or without 10 μM erlotinib for 1 h. To study the anti-HBV effects of EGFR in HBV-infected model, HepG2-NCTP cells infected with HBV (2✕10² GEq/cells) for 6 days before treating with or without 2.5 μM erlotinib, 10 μM gefitinib or 30 nM ETV (Sigma-Aldrich Corporation) for 48 h to insure the effect evaluation at the summit of HBV replication. To study the sustained influences of EGFR inhibitors on viral antigens and the cccDNA reservoir, HepG2-NCTP cells were treated with or without 2.5 μM erlotinib for up to 8 days. erlotinib, gefitinib and ETV were all dissolved in dimethylsulfoxide (DMSO). The solvent was standardized at the concentration of 0.125% (V/V).