Extended Data Fig. 8: ADAR1 depletion causes ZBP1-dependent cell death of human HT-29 cells.
From: ADAR1 prevents autoinflammation by suppressing spontaneous ZBP1 activation
a, Immunoblot of HT-29 cells 48 h post transfection with non-targeting control siRNAs (si-CTRL), siRNAs targeting ADAR (si-ADAR) or the p150 isoform of ADAR (si-ADAR-p150). As controls, cells were treated with 1,000 U/mL IFN-α or 100 ng/mL IFN-γ. b, HT-29 cells stably expressing wild type ZBP1 (ZBP1WT) or Zα domain mutant ZBP1 (ZBP1Zα1Zα2mut) were transfected with non-targeting control siRNAs (si-CTRL) or siRNAs targeting ADAR (si-ADAR). Cell death was quantified by measuring relative (rel.) SYTOX Green uptake every 2 h. c, HT-29 ZBP1WT and ZBP1Zα1Zα2mut cells were treated with 30 ng/mL human TNF, 5 µM BV6 and 20 µM zVAD-fmk (TNF + BV6/ZVAD) or 30 ng/mL TNF and 20 µg/mL CHX (TNF + CHX). Cell death was quantified as in (b). d, Immunoblot related to (c). Cells were harvested 3 (TNF + BV6/ZVAD) or 6 (TNF+CHX) hours post treatment. FL, full length. e, HT-29 ZBP1WT cells were transfected with si-ADAR only or si-ADAR and si-ZBP1. After five hours, cells were treated with 20 µM zVAD-fmk and/or 3 µM GSK’840 or left untreated. Cell death was analysed as in (b). f, Immunoblot related to (e). Samples were harvested 30 h post transfection. TNF + CHX and TNF + BV6/ZVAD control samples were treated and harvested as in (d). g, HT-29 ZBP1WT cells were transfected with si-CTRL or si-ADAR-p150. si-ADAR-p150 was combined with si-ZBP1, or siRNAs targeting RIPK1 (si-RIPK1), RIPK3 (si-RIPK3), TICAM1 (TRIF; si-TICAM1) or CASP8 (si-CASP8). Cell death was quantified as in (b). h, HT-29 ZBP1WT cells transfected with the indicated siRNAs were harvested at 48 h post transfection for protein expression analysis. Fitted lines in (b,c,e) represent a logistic growth fit; Data points show mean of 3 (b,c,e) or 4 (g) technical replicates + SD and are representative of 3 independent experiments. For gel source data, see Supplementary Figure 1
