The human cell lines: HCC827 (ATCC® CRL-2868™) and HCC4006 (ATCC® CRL-2871™), kindly provided by Oreste Segatto, were cultured in RPMI 1640 medium (BioWhittaker, Lonza, USA) supplemented with 10 mM Hepes pH 6.98–7.30, 1 mM L-glutamine, 100 U/ml penicillin/streptomycin (BioWittaker, Lonza) and heat inactivated 10% fetal bovine serum (FBS) (Sigma-Aldrich). All cells were cultured at 37°C in a 5% CO2 humidified incubator. Erlotinib (ERL)-resistant cell lines (RA1, RA2, RB1, RB1.1, RB2 and RC2.2) established essentially as previously described [29 (link)] were cultured in the same experimental conditions. Briefly, HCC827 and HCC4006 parental cell lines were cultured in complete tissue culture medium with a stepwise increase of erlotinib concentrations (stepwise method), or a high concentration of erlotinib (1 μM) (high-concentration method) over 5–6 months.
The primary antibodies: EGFR (clone D09, kindly provided by O. Segatto); phosphorylated-EGFR (Tyr1068); HER2/ErbB2 (D8F12); HER3/ErbB3 (D22C5); HER4/ErbB4 (111B2); phospho-HER2/ErbB2 (Y1221/1222) (6B12); phospho-HER3/ErbB3 (Y1289) (D1B5); phospho-HER4/ErbB4 (Y1284) (21A9); p44-42 MAPK (ERK1/2) (#9102), phospho-p44-42 MAPK (ERK1/2) (T202/Y204) clone E10; c-MET (D1C2); phospho-c-MET (Y1234/1235) (D26); Akt and phospho-Akt (S473), clone D9E were from Cell Signaling Technology (CST); AXL (#AF154) and phospho-AXL (Y779) (#AF228) were from R&D Systems; GAPDH, clone 1D4 or #5174 was respectively from Novus Biologicals or CST. Secondary antibodies: goat anti-rabbit IgG (H+L)-HRP and goat anti-mouse IgG (H+L)-HRP were from Bio-Rad; donkey anti-goat IgG-HRP (sc 2020) was from Santa Cruz Biotechology; streptavidin Alexa Fluor-488 were from Life Technologies. Biotin-labeled horse anti-rabbit Ig was from Vector Laboratories Burlingame, CA, USA. Tyrosine kinase inhibitors: Erlotinib Hydrochloride Salt, gefitinib Free Base, Vandetanib Free Base Lapatinib, Imatinib and Paclitaxel were from LC Laboratories, USA; SU11274, PHA-665752 hydrate and PF-04217903 were from Sigma-Aldrich, AZD9291, Rociletinib (CO-1686, AVL-301) and R428 (BGB324) were from Selleckchem and distributed by DBA Italia. Stock solutions of 10 mM for all inhibitors were prepared in DMSO and stored at -20°C or -80°C. MTT, 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was from Sigma-Aldrich. MTT stock solution (5 mg/ml in H2O, sterilized by filtration) was stored at 4°C for 1 months. Power SYBR Green PCR Master Mix was from Applied Biosystems. TRIzol reagent was from Life Technologies, Reverse Transcription System was from Promega.
The primary antibodies: EGFR (clone D09, kindly provided by O. Segatto); phosphorylated-EGFR (Tyr1068); HER2/ErbB2 (D8F12); HER3/ErbB3 (D22C5); HER4/ErbB4 (111B2); phospho-HER2/ErbB2 (Y1221/1222) (6B12); phospho-HER3/ErbB3 (Y1289) (D1B5); phospho-HER4/ErbB4 (Y1284) (21A9); p44-42 MAPK (ERK1/2) (#9102), phospho-p44-42 MAPK (ERK1/2) (T202/Y204) clone E10; c-MET (D1C2); phospho-c-MET (Y1234/1235) (D26); Akt and phospho-Akt (S473), clone D9E were from Cell Signaling Technology (CST); AXL (#AF154) and phospho-AXL (Y779) (#AF228) were from R&D Systems; GAPDH, clone 1D4 or #5174 was respectively from Novus Biologicals or CST. Secondary antibodies: goat anti-rabbit IgG (H+L)-HRP and goat anti-mouse IgG (H+L)-HRP were from Bio-Rad; donkey anti-goat IgG-HRP (sc 2020) was from Santa Cruz Biotechology; streptavidin Alexa Fluor-488 were from Life Technologies. Biotin-labeled horse anti-rabbit Ig was from Vector Laboratories Burlingame, CA, USA. Tyrosine kinase inhibitors: Erlotinib Hydrochloride Salt, gefitinib Free Base, Vandetanib Free Base Lapatinib, Imatinib and Paclitaxel were from LC Laboratories, USA; SU11274, PHA-665752 hydrate and PF-04217903 were from Sigma-Aldrich, AZD9291, Rociletinib (CO-1686, AVL-301) and R428 (BGB324) were from Selleckchem and distributed by DBA Italia. Stock solutions of 10 mM for all inhibitors were prepared in DMSO and stored at -20°C or -80°C. MTT, 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was from Sigma-Aldrich. MTT stock solution (5 mg/ml in H2O, sterilized by filtration) was stored at 4°C for 1 months. Power SYBR Green PCR Master Mix was from Applied Biosystems. TRIzol reagent was from Life Technologies, Reverse Transcription System was from Promega.
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