1�C0.2mM (Chawla et al, 1984; Gmunder et al, 1991). Therefore, inhibitor we defined the normal cystine concentration to be 0.1mM cystine. Accordingly, cystine concentrations lower than 0.1mM or higher than 0.2mM were considered as low or high cystine concentrations, respectively. Cell survival/proliferation assay Cell proliferation and viability were determined by neutral red uptake (Babich and Borenfreund, 1991). Cells were plated in 96-well plates at 1000 cells per well. The next day, cells were treated as dictated by the particular experiments. Wells were then aspirated and incubated with 100��l 0.0025% neutral red dye in cell culture medium. Empty wells were also incubated with neutral red dye to allow for background absorbance correction.
After 4h of incubation, wells were aspirated and 100��l 1% acetic acid in 50% ethanol was added per well to solubilise intracellular neutral red dye. Absorbance was determined at 550nm using a 96-well VERSA max microplate reader (Molecular Devices, Sunnyvale, CA, USA). RNA isolation and q-RT�CPCR Isolation of total RNA from cultured cells in vitro or tumour cells in vivo was performed using an RNeasy Micro kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s recommendations. RNA (5��g) was used to synthesise first-strand cDNA using the Superscript II RT�CPCR kit (Invitrogen), according to the manufacturer’s recommendations. q-RT�CPCRs were performed in 384-well plates using SYBR? Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) in a volume of 15��l (containing 50ng of cDNA).
Fluorescence emission was detected for each PCR cycle on a ABI Prism? 7900 Sequence Detection System (Applied Biosystems) and threshold cycle (Ct) values were determined based on a 40-cycle reaction. Thermal cycling conditions were 50��C for 2min and 95��C for 5min, followed by 40 cycles of 15s at 95��C, 30s at 58��C, and 30s at 72��C. Average Ct values from duplicate PCRs were normalised to average Ct values for the housekeeping gene ��-2-microglobulin (��2M) from the same cDNA preparations. The relative mRNA expression of a gene was calculated as follows: Average Ct (��2M)?Average Ct (gene)=d; mRNA expression relative to ��2M=2^(d). Each q-RT�CPCR was carried out in duplicate.
Primer sequences are as follows: mouse 4F2hc: Dacomitinib forward 5��-GAGGACAGGCTTTTGATTGCAG-3��, reverse 5��-AGGTAGGAGCTGGTCAACAGCA-3��; mouse xCT: forward 5��-GAGGACAGGCTTTTGATTGCAG-3��, reverse 5��-AGGTAGGAGCTGGTCAACAGCA-3��; mouse ��2M: forward 5��-CACCCCCACTGAGAGACTGATACA-3��, reverse 5��-TGATGCTTGATCACATGTCTCG-3��; human 4F2hc: forward 5��-AGTGCCAACATGACTGTGAAG-3��, reverse 5��-CCTTACTCCGCTGGTCACTCAG-3��; human xCT: forward 5��-TGCTGGCTGGTTTTACCTCAAC-3��, reverse 5��-CCAATGGTGACAATGGCCAT-3��; human ��2M: forward 5��-ACCATGTGACTTTGTCACAGCC-3��, reverse 5��-AATCCAAATGCGGCATCTTC-3��.