Fig. 3

Loss of SLC39A7 mediates resistance to TNFα-induced cell death by diminishing TNFR1 surface expression. a Growth curve for KBM7 FADD- sgRen or SLC39A7- cells. Equal cell numbers were seeded and cell growth monitored over 7 days. Data represent mean value ± s.d. of five independent experiments; statistical analysis by t-test, *P < 0.01. b Cell viability in KBM7 FADD- sgRen or SLC39A7- cells treated overnight (16 h) with TNFα (10 ng/ml), SMAC mimetic (0.5 µM), and Nec-1s (50 µM) as indicated. Cell viability was assessed using a luminescence-based readout for ATP (CellTiter Glo). Data represent mean value ± s.d. of two independent experiments performed in triplicates. c KBM7 FADD- SpCas9 (empty, sgRen or SLC39A7-) cells were stimulated for the time indicated with TNFα (10 ng/ml). Cells were then lysed and subjected to immunoblotting with the indicated antibodies. d Flow cytometry analysis for TNFR1 surface expression. KBM7 TNFRSF1A- cells serve as negative control for background staining. Data shown are representative of two independent experiments. e KBM7 FADD- SpCas9 (empty, sgRen or SLC39A7-) cells were treated for 7 h with Brefeldin A (0.5 µM), Tunicamycin (2 µM), Thapsigargin (0.5 µM), MG-132 (10 µM) or DMSO as control. Cells were then lysed and subjected to immunoblotting with the indicated antibodies. f KBM7 TNFRSF1A-, KBM7 FADD- and KBM7 FADD- SLC39A7- cells were lysed and subjected to immunoblotting with the indicated antibodies. g KBM7 FADD- SpCas9 sgRen or SLC39A7- cell lysates were incubated for 1 h at 37 °C in presence or absence of PNGaseF or EndoH, respectively, and analysed by immunoblot with the indicated antibodies. Immunoblots shown are representative of two independent experiments. Asterisk (*) indicates non-specific band