Nci h1993

Experiments were carried out with human lung adenocarcinoma cell lines A549 (ATCC CCL_185), Calu6 (ATCC HTB-56) and NCI-H1993 (ATCC CRL-5909). All cell lines were obtained from American Type Cell Culture Collection (ATCC) and cultured in DMEM growth medium (GIBCO #31966-21), supplemented with 10% heat-inactivated foetal bovine serum (FBS) and 1% antibiotics (penicillin, streptomycin). Cells were kept at 37 °C and 5% CO₂ in the incubator and were passaged at 80–90% confluence every 2–3 days to maintain continuous logarithmic growth. All cell lines studied in this work were erlotinib resistant and EGFR wild type (Table 1). Cells were treated with pitavastatin calcium (SelleckChem, #S1759), fluvastatin sodium (SelleckChem, #S1909) and erlotinib hydrochloride (SelleckChem, #S1023).Table 1

Human NSCLC cell lines harbouring different genetic mutations examined in the study.

Cell line	EGFR	K-Ras	Met	p53	Erlotinib
A549	Wildtype	Mutated	Wildtype	Wildtype	Resistant
Calu6	Wildtype	Mutated	Wildtype	Mutated	Resistant
H1993	Wildtype	Wildtype	Amplified	Mutated	Resistant

This study was approved by the Ethics Committee of Sun Yat-sen University Cancer Center. The human NSCLC cell lines NCI-H292 (ATCC CRL-1848), NCI-H460 (ATCC HTB-177), PC-9 (RRID:CVCL_B260), A549 (ATCC CCL-185), NCI-H1650 (ATCC CRL-5883), NCI-H1993 (ATCC CRL-5909), NCI-H1975 (ATCC CRL-5908), HCC827 (ATCC CRL-2868), and NCI-H1299 (ATCC CRL-5803) were obtained from the State Key Laboratory (SKL) of Oncology in South China. These cells grew at 37 °C in a humidified atmosphere of 95% air and 5% CO2 using Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum.

All human NSCLC cell lines (NCI-H1993, A549, NCI-H358, NCI-H1975, NCI-H1650, HCC827) used in this study were purchased from American Type Culture Collection (Manassas, VA, USA). These NSCLC cell lines were maintained in RPMI-1640 (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) supplemented with 5% fetal bovine serum (FBS) and cultured at 37 °C in a humidified atmosphere containing 5% CO₂. Antibodies used in western blotting were purchased from the following companies: anti-ERK1/2 (M5670, rabbit, Sigma-Aldrich, Merck KGaA); anti-phospho-ERK1/2 (Thr202/ Tyr204) (9101S, rabbit, Cell Signaling Technology, Danvers, MA, USA); anti-Cadherin (ab15148, rabbit, Abcam, Cambridge, MA, USA); anti-Vimentin (ab92547, rabbit, Abcam); anti-Actin (ab3280, mouse, Abcam). Reagents used in the study were from the following companies: human recombinant TGFβ1 (T7039, Sigma Aldrich, Merck KGaA), human recombinant VEGF-C (SRP3184, Sigma Aldrich, Merck KGaA), and LY2157299 (S2230, Selleckchem, Houston, TX, USA).

A549, NCI-H1993, NCI-H226, NCI-H441 human lung adenocarcinoma cells, Hs746T human gastric carcinoma cells, Caki-I kidney carcinoma cells, and 293T embryonic kidney cells were from the American Type Culture Collection (ATCC/LGC Standards Srl). EBC-1 human lung carcinoma cells were from the Japanese Collection of Research Bioresources. GTL-16 is a clone derived from MKN-45 human gastric carcinoma cells [27 (link)]. GTL-16_Res were obtained by prolonged treatment of GTL-16 cells with the anti-MET Tyrosine Kinase Inhibitor (TKI) PHA-665752 [28 (link)]. Cell lines were cultured according to the manufacturer’s instructions. GTR-164, GTR-498, and GTR-210 primary gastric carcinoma cells were derived from the respective Patient Derived Xenografts and maintained as described in [29 (link)]. L1.13 Cancer of Unknown Primary origin (CUP) primary cell line was derived and maintained as described in [15 (link)]. Huvec cells were obtained from dr. Gabriella Doronzo (University of Turin) and maintained as described in [30 (link)]. Primary human skin fibroblasts were obtained from Prof. Antonia Follenzi (University of Piemonte Orientale) and maintained as described in [31 ]. Primary human epidermal keratinocytes were from Lonza and maintained as suggested by the supplier.