Titles and legends to supplemental figures Figure S1 Overexpression of ARC suppresses TNF-induced programmed necrosis. (a) TNF-induced LDH release is inhibited by ARC overexpression in L929 cells. LDH release is not stimulated by TNFα + cycloheximide (TNFα+CHX) treatment. Data shown as mean ± S.E., n = 3. ****P-value < 0.0001 versus empty vector. (b) ARC overexpression suppresses PI entry into cells following treatment of cells with TNFα. Mean ± S.E. from n = 8. *P-value< 0.05 versus empty vector.
Figure S2 The ARC CARD is required for the ability of ARC to suppress TNF-induced necrosis. TNFα–induced PI entry is suppressed by ARC, but not by the CARD-defective ARC mutant. Means ± S.E. from n = 3 experiments. **P-value < 0.01 versus empty vector.
Figure S3 The knockdown of ARC by a second hairpin also sensitizes L929 cells to TNF-induced LDH release. Left panel. Immunoblot of lysates from L929 cells stably transfected with scrambled shRNA (Scr) or a second shRNA against ARC (sh15) showing efficient knockdown of ARC. Right panel. TNF-induced LDH release in these cell lines showing that knockdown of ARC with the second hairpin augments TNF-induced necrosis. Necrostatin-1 (Nec-1) is a small molecule inhibitor of the RIP1 kinase activity and death receptor-mediated necrosis. Data shown as mean S.E. from n = 3. ****P-value < 0.0001 compared with scrambled shRNA.
Figure S4 The knockdown of ARC sensitizes MCF7 cells overexpressing RIP3 to TNF-induced LDH release. (a) Immunoblot showing that MCF7 cells express higher levels of endogenous ARC compared to other cell lines shown. (b) Immunoblot showing the levels of RIP3 expression and ARC knockdown in different MCF7 cell lines generated. (c) TNF-induced LDH release in these cell lines showing that knockdown of ARC augments TNF-induced necrosis in MCF7 cells. TNF + CHX + zVAD induces necrosis. Data shown as mean S.E. from n = 3. ****P-value < 0.0001 compared with scrambled shRNA overexpressing RIP3.
Figure S5 Overexpression of ARC inhibits TNF-induced necrosis independently of TRADD. Top panel. Immunoblot showing TRADD knockdown with siRNA in L929 cells stably transduced with empty vector (Φ) or ARC-HA. Bottom panel. LDH release in response to vehicle, TNFα alone, or TNFα + necrostatin-1 (Nec-1) for 12 hours. LDH release occurs as effectively in cells in which TRADD is present or depleted (Empty; Scr versus Empty; siTRADD, and ARC-HA;Scr versus ARC-HA;siTRADD, P-value = ns). n = 5 independent experiments. **P-value < 0.01 (ARC-HA; Scr compared with Empty; Scr and ARC-HA; siTRADD compared with Empty; siTRADD).
Figure S6 Proposed model of the mechanism by which ARC suppresses TNF-induced necrosis through disrupting complex I formation. The ligation of TNF to TNF receptor 1 (TNFR1) stimulates the formation of complex I consisting of TNF receptor associated death domain protein (TRADD) and receptor interacting protein kinase 1 (RIP1). Complex I subsequently signals to activate NF-B survival pathway or to initiate the execution of necrosis or apoptosis. However, when present, ARC interacts directly with TNFR1 to prevent the recruitment of RIP1 and TRADD, thereby blocking TNF-induced NF-B activation, necrosis, and apoptosis.