TNF-Induced Activation of the Nox1 NADPH Oxidase and Its Role in the Induction of Necrotic Cell Death  You-Sun Kim, Michael J. Morgan, Swati Choksi, Zheng-gang Liu  Molecular Cell  Volume 26, Issue 5, Pages 675-687 (June 2007) DOI: 10.1016/j.molcel.2007.04.021 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 BHA Prevents TNF-Induced O2− Generation and Necrotic Cell Death (A) NADPH oxidase assays in TNF-treated and untreated L929 cells. Time of TNF treatment is indicated. (B) NADPH oxidase assays in p65−/− MEFs. (C and D) Cell death assays (MTT). Cells were untreated, or pretreated for 30 min with BHA and/or zVAD and then treated with TNF for the indicated times (error bars, ±SEM). (E) NADPH oxidase assays in TNF-treated and untreated L929 cells in the presence or absence of BHA. Molecular Cell 2007 26, 675-687DOI: (10.1016/j.molcel.2007.04.021) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 The NADPH Oxidase Nox1 Is Expressed in L929 Cells and Is Responsible for TNF-Induced O2− Generation (A) Cell lysates from various cell lines blotted with anti-gp91 (Nox2), anti-Nox1, and anti-actin antibodies. (B) S-35 autoradiography of in vitro translated untagged (top) or Xpress-tagged (bottom) plasmid constructs of Nox1. In vitro translated p51NOXA1 and p47phox are shown for size comparison in addition to the indicated molecular markers. (C) Myc-Nox1 immunoprecipitated with an anti-mouse Myc antibody from transfected 293 cells and blotted with anti-Nox1 antibody. (D) Western blot of L929 cells transfected with Nox1-specific siRNA (#4 and #2) or control siRNA (Lamin A/C) 72 hr posttransfection. (E) NADPH oxidase assays in cells from (D). For clarity purposes luminescence data are now shown as the RLU ratio between the TNF-treated and untreated samples and indicate the relative fold activation of NADPH oxidase by TNF. (F) NADPH oxidase assays from cells cotransfected with Nox1 #4 siRNA oligo along with either a human Nox1 expression plasmid, an inactive point mutant (T341K), or empty vector. Molecular Cell 2007 26, 675-687DOI: (10.1016/j.molcel.2007.04.021) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 TRADD, RIP1, and Rac1 Form a Complex with Nox1/NOXO1 Following TNF Treatment (A) Western blots of endogenous L929 cell protein coimmunoprecipitated by anti-TRADD antibody from TNF-treated cells for the indicated times. (B) Western blots showing immunoprecipitation (IP) experiments in 293 cells overexpressing proteins from Xp-His, Xp-His-NOXO1, or Xp-His-NOXA1 plasmids in combination with either Flag-RIP1 (left) or Flag-TRADD (right). Due to the differences in binding affinity, the RIP1 immunoprecipitation was carried out for 5 hr after lysis, while the TRADD immunoprecipitation was done overnight. (C) Western blots of anti-NOXO1 antibody precipitates of endogenous protein from untreated L929 cells or after TNF treatment. Lanes were cropped where indicated to simplify the figure. (D) Immunoprecipitation of endogenous protein from wild-type, RIP−/−, and TRAF2−/− MEFs using the anti-TRADD antibody. Cells were pretreated with CHX and zVAD for 30 min before TNF treatment or left untreated for the control. Molecular Cell 2007 26, 675-687DOI: (10.1016/j.molcel.2007.04.021) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Interaction of NOXO1 with TRADD Requires Its Polyproline Region (A and B) Western blots of immunoprecipitation (IP) experiments performed in 293 cells transfected with the Flag, Flag-TRADD, or Flag-TRADD P-A mutant (TRADD prolines 192–198 changed to AAAAAGT) plasmids and (A) Xpress-His Vector or Xp-His-NOXO1 plasmids, (B [top]) Myc or Myc-RIP1 plasmids, (B [bottom left]) GFP-FADD plasmid, or (B [bottom right]) HA-TRAF2 plasmid. (C) Immunoprecipitation of His-tagged proteins from 293 cells cotransfected with Flag or Flag-TRADD along with Xpress-His Vector, Xp-His-NOXO1, or Xp-His-NOXO1 SH3 mutant plasmids. Molecular Cell 2007 26, 675-687DOI: (10.1016/j.molcel.2007.04.021) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Effect of Stable Expression of TRADD P-A Mutant on TNF-Induced O2− Generation and Cell Death (A) Expression levels of TRADD and TRADD P-A mutant in L929 stable cell lines. Similar protein amounts were added. (B) Western blots showing intact JNK and IκBα degradation pathways in Flag-vector, Flag-TRADD, and Flag-TRADD P-A mutant stable cell lines from (A) in response to TNF. (C) TRADD immunoprecipitation experiments in TRADD and TRADD P-A mutant stable cell lines. (D) NADPH oxidase assays in cells from (A). The luminescence ratio is shown indicating the relative fold activation of NADPH oxidase by TNF. (E) Cell viability data of stable cell lines from (A) treated for TNF for 10 hr and analyzed by MTT assay data (error bars, ±SEM) (left) or phase-contrast microscopy (right). Molecular Cell 2007 26, 675-687DOI: (10.1016/j.molcel.2007.04.021) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Effect of Dominant-Negative Rac1 in TNF-Induced O2− Generation and Cell Death (A) Expression levels of the N17-Rac1 mutant in L929 stable cell lines. (B) Western blots showing intact JNK and IκBα degradation pathways in Flag-vector or Flag-N17-Rac1 mutant stable cells from (A) in response to TNF at the indicated times. (C) NADPH oxidase assays in cells from (A). The luminescence ratio is shown indicating the relative fold activation of NADPH oxidase by TNF. (D) MTT assay of stable cell lines from (A)–(C) treated with TNF for 10 hr (error bars, ±SEM). (E) Viability assays (MTT) of L929 cells transfected with Nox1-specific siRNA (#4 and #2) or control siRNA (Lamin A/C) and treated with TNF. As shown in Figure 2D, little Nox1 reduction was seen in cells transfected with Nox1 oligo #2. Representative phase-contrast microscopy images (top) and MTT assay data at 24 hr (error bars, ±SEM) (bottom) are shown. (F) MTT assay of L929 cells cotransfected with Nox1 #4 siRNA oligo and either a human Nox1 expression plasmid, an inactive point mutant (T341K), or empty vector and treated with TNF for 24 hr (error bars, ±SEM). Molecular Cell 2007 26, 675-687DOI: (10.1016/j.molcel.2007.04.021) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Prolonged JNK Activation Is Involved in TNF-Induced O2 Generation and Cell Death (A and B) Time course of JNK phosphorylation in (A) Flag-TRADD and Flag-TRADD P-A mutant L929 stable cell lines or (B) Flag or Flag-N17-Rac1 mutant L929 stable cells. (C) Cell viability after TNF treatment (8 hr) as determined by MTT assay (error bars, ±SEM) in L929 cells pretreated (30 min) with BHA or SP600125, an inhibitor of JNK. (D) Schematic diagram of proposed pathway. Molecular Cell 2007 26, 675-687DOI: (10.1016/j.molcel.2007.04.021) Copyright © 2007 Elsevier Inc. Terms and Conditions