These studies used CHO WTP4 cells, which are not killed by LF, and the cytotoxic fusion protein FP59 [27], [27], in which the N-terminus of LF is fused to the ADP-ribosylating domain of Pseudomonas exotoxin A. CHO cells were maintained in alpha minimal essential medium supplemented with 5% fetal bovine serum, 2 mM glutamine, 50 µg of gentamicin/ml, and 25 mM HEPES.
This cell line was maintained in alpha minimum essential medium (α-MEM) supplemented with 10% (FBS) (Invitrogen, Frederick, MD) plus a penicillin/streptomycin cocktail.
SK-MEL-1 was maintained in alpha modified Eagle's Minimal Essential Media (MEM) supplemented with 10% fetal bovine serum (FBS), and Daudi and LNCaP were maintained in RPMI-1640 Media supplemented with 10% FBS.
Cells were maintained in alpha minimal essential medium (Gibco-BRL,Gaithersburg, MD, USA) supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) normal chicken serum, 100 µmol/l β-mercaptoethanol (Sigma-Aldrich, St Louis, MO, USA), 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco-BRL), at a maximum density of 1 × 106 cells per ml.
Human NK-92 cell line was purchased from the American Type Culture Collection (ATCC CRL-2407, Rockville, MD) and maintained in alpha MEM medium supplemented with 12.5% horse serum, 12.5% fetal bovine serum (FBS), 0.2 mM myoinositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, and recombinant human interleukin-2 (Proleukin, Prometheus, San Diego, CA).
Mouse embryonal carcinoma P19 cells were used in this study, and RA-induced P19 cell neuronal differentiation was performed with a slight modification of procedures described by Lee et al. Briefly, P19 cells were maintained in alpha minimum essential medium (α-MEM; Invitrogen) supplemented with 7.5% bovine serum (BS; Invitrogen) and 2.5% FBS at 37 °C in a humidified atmosphere (5% CO2 and 95% air).
EMT6 cells were maintained in alpha-minimal essential medium (Mediatech, Manassas, VA) supplemented with 10% FBS.
P19 cells (ATCC) were maintained in alpha-MEM with 7.5% calf serum, 2.5% fetal bovine serum, 2 mM L-glutamine and antibiotics.
The rat mammary adneocarcinoma line MTLn3 was maintained in alpha-MEM supplemented with 10% fetal bovine serum, 1% L-glutamine and 1% penicillin-streptomycin. Transient transfections were conducted with 3×106 cells and two micrograms of plasmid construct or siRNA using the Nucleofector I device (Amaxa Biosystems, Berlin, Germany).
The human OS cell lines U2OS (ATCC # HTB-96) and MG63 (ATCC CRL-142727 ) were purchased from American Type Culture Collection ATCC (Rockville, MD) and maintained in alpha-Minimum Essential Medium (alpha-MEM) supplemented with 10% heat inactivated Fetal Bovine Serum and 2 mM L-Glutamine.
NK-92 cells were maintained in alpha-MEM containing 12.5% DES, 12.5% FBS, and 10 ng/mL interleukin-2.
