UOK262 cell lines were a gift from Dr. Marston Linehan, National Cancer Center, National Institute of Health.(13 (link)) NCCFH1 was previously developed from the pleural fluid of a patient with metastatic HLRCC.(14 (link)) UOK262 cell line was authenticated through Sanger’s sequencing to confirm the presence of c.1316A>C FH mutation. NCCFH1 cell line was authenticated in the same way to confirm the presence of c.1162 delA FH mutation. All HLRCC cells were maintained in RPMI1640 media (Gibco, cat # 11875–093) with 10% Fetal Bovine Serum (Genesee Scientific). Both cell lines have been tested negative for mycoplasma contamination using a QPCR based method.
For cysteine deprivation experiments, L-glutamine, L-methionine and L-cystine free RPMI1640 media (Sigma, Cat # R7513) were used instead. The media was back supplemented with L-glutamine (Sigma, Cat # G7513) to a final concentration of 2mM, L-methionine (Sigma, Cat # M5308) to a final concentration of 0.1mM, and either without L-cystine supplementation for cysteine deprivation group, or with L-cystine hydrochloride (Sigma, Cat # 57579) supplementation to a final concentration of 0.2mM for the control group. Cysteine deprivation experiments were carried out in the presence of 5% instead of 10% FBS to minimize the effects of cysteine present in the Fetal Bovine Serum.
For cysteine deprivation experiments, L-glutamine, L-methionine and L-cystine free RPMI1640 media (Sigma, Cat # R7513) were used instead. The media was back supplemented with L-glutamine (Sigma, Cat # G7513) to a final concentration of 2mM, L-methionine (Sigma, Cat # M5308) to a final concentration of 0.1mM, and either without L-cystine supplementation for cysteine deprivation group, or with L-cystine hydrochloride (Sigma, Cat # 57579) supplementation to a final concentration of 0.2mM for the control group. Cysteine deprivation experiments were carried out in the presence of 5% instead of 10% FBS to minimize the effects of cysteine present in the Fetal Bovine Serum.
