Journal: Cell Death and Differentiation

Article Title: Combination of IAP antagonist and IFN γ activates novel caspase-10- and RIPK1-dependent cell death pathways

doi: 10.1038/cdd.2016.147

Figure Lengend Snippet: Synergistic cell death induced by IFNγ/SM occurs via RIPK3-dependent- and caspase-8 mediated apoptosis in murine fibroblasts. (a) Wild-type, Ripk3−/−, Mlkl−/−, Ripk3−/−Casp8−/− and Mlkl−/−Casp8−/− MDFs were treated with IFNγ/SM or not treated (UT) for 48 h. Cell death was measured by detecting PI-permeable cells using flow cytometry. Data are plotted as mean±S.E.M. (n≥3). (b and c) MDFs similar to that in (a) were treated with 1 μM Compound 1 (MLKL inhibitor), 50 μM Nec-1 and IFNγ/SM without (b) or with (c) 10 μM QVD (Q-VD-OPh) or not treated (UT) for 48 h. The same concentrations were used throughout the paper. Cell death was measured by detecting PI-permeable cells using flow cytometry. Data are represented as mean±S.E.M. (n≥3). (d) Wild-type, Mlkl−/−, Ripk3−/−, Ripk3−/−Casp8−/− and Tnfr1−/− MDFs were treated with IFNγ/SM, QVD and Nec-1 for 24 h similar to that in (b) and (c). Total cell lysates were analysed by immunoblotting (n=2). Arrows indicate full-length and processing products of caspases

Article Snippet: Antibodies used for immunoblotting were as follows: anti-human FADD (BD Pharmingen, North Ryde, NSW, Australia; 556402), anti-human FLIP (Enzo Life Sciences, Redfern, NSW, Australia; ALX-804-961-0100), anti-full-length mouse caspase-8 (Enzo Life Sciences; ALX-804-448-C100), anti-cleaved mouse caspase-8 (Cell Signalling Technology, Danvers, MA, USA; 8592), anti-cIAP1 and anti-cIAP2 (Alexis Biochemicals, San Diego, CA, USA; ALX-803-341), anti-XIAP (MBL, M044-3), anti-FLAG M2 (Sigma; F-3165), anti-β-actin (Sigma; A-1978), anti-mouse caspase-8 (Cell Signalling Technology; 4927), anti-human caspase-8 (MBL, Woburn, MA, USA; M058-3), anti-caspase-10 (MBL; M059-3), anti-cleaved caspase-3 (Cell Signalling Technology; 9661), anti-human PKR (Santa Cruz; sc6282), anti-RIPK1 (BD Transduction Laboratories, North Ryde, NSW, Australia, 610458), anti-mouse RIPK3 (Axxora, Farmingdale, NY, USA; PSC-2283-c100), anti-human phospho-MLKL (Abcam, Milton, Cambridge, UK; ab187091), anti-total mouse and human MLKL (housemade, 3H1).

Techniques: Flow Cytometry, Western Blot

Journal: Cell Death and Differentiation

Article Title: Combination of IAP antagonist and IFN γ activates novel caspase-10- and RIPK1-dependent cell death pathways

doi: 10.1038/cdd.2016.147

Figure Lengend Snippet: Inhibitors of apoptosis and necroptosis are unable to completely protect HT29 cells from IFNγ/SM-induced killing. (a) HT29, D645 and KATOIII cells were treated with IFNγ/SM, QVD and Nec-1 or not treated (UT) as indicated for 48 h. Cell death was measured by detecting PI-permeable cells using flow cytometry. Data are plotted as mean±S.E.M. (n≥3). (b, and c) Wild-type (black bar) and CRISPR/Cas9 CASP8−/− (white bar; b) and RIPK1−/− (red bar), RIPK3−/− (blue bar), MLKL−/− (green bar; c) HT29 cells were treated with IFNγ/SM, QVD and Nec-1 or left untreated (UT) as indicated for 48 h. Cell death was meaured by detecting PI-permeable cells using flow cytometry. Data are plotted as mean±S.E.M. (n≥3). (d) Immunoblot of wild-type, CRISPR/Cas9 MLKL−/−, RIPK3−/− and RIPK1−/− HT29 cells treated with IFNγ/SM, QVD and Nec-1 or not treated for 24 h. Arrows indicate full-length and processing products of caspases

Article Snippet: Antibodies used for immunoblotting were as follows: anti-human FADD (BD Pharmingen, North Ryde, NSW, Australia; 556402), anti-human FLIP (Enzo Life Sciences, Redfern, NSW, Australia; ALX-804-961-0100), anti-full-length mouse caspase-8 (Enzo Life Sciences; ALX-804-448-C100), anti-cleaved mouse caspase-8 (Cell Signalling Technology, Danvers, MA, USA; 8592), anti-cIAP1 and anti-cIAP2 (Alexis Biochemicals, San Diego, CA, USA; ALX-803-341), anti-XIAP (MBL, M044-3), anti-FLAG M2 (Sigma; F-3165), anti-β-actin (Sigma; A-1978), anti-mouse caspase-8 (Cell Signalling Technology; 4927), anti-human caspase-8 (MBL, Woburn, MA, USA; M058-3), anti-caspase-10 (MBL; M059-3), anti-cleaved caspase-3 (Cell Signalling Technology; 9661), anti-human PKR (Santa Cruz; sc6282), anti-RIPK1 (BD Transduction Laboratories, North Ryde, NSW, Australia, 610458), anti-mouse RIPK3 (Axxora, Farmingdale, NY, USA; PSC-2283-c100), anti-human phospho-MLKL (Abcam, Milton, Cambridge, UK; ab187091), anti-total mouse and human MLKL (housemade, 3H1).

Techniques: Flow Cytometry, CRISPR, Western Blot

Journal: Cell Death and Differentiation

Article Title: Combination of IAP antagonist and IFN γ activates novel caspase-10- and RIPK1-dependent cell death pathways

doi: 10.1038/cdd.2016.147

Figure Lengend Snippet: HT29 cells deficient for caspase-8-mediated apoptosis and necroptosis remain sensitive to IFNγ/SM-induced killing. (a) Wild-type and CRISPR/Cas9 RIPK1−/−CASP8−/− (red frame), RIPK3−/−CASP8−/− (blue frame) and MLKL−/−CASP8−/− (green frame) HT29 cells were treated with IFNγ/SM, QVD and Nec-1 or left untreated (UT) as indicated for 48 h. Cell death was analysed by measuring PI-permeable cells using flow cytometry. Data are plotted as mean±S.E.M. (n≥3). (b) Wild-type and CRISPR/Cas9 CASP8−/−, RIPK1−/−CASP8−/−, RIPK3−/−CASP8−/−, MLKL−/−CASP8−/− HT29 cells were treated with IFNγ/SM, QVD and Nec-1 or not treated for 24 h before SDS lysis, separation on SDS-PAGE and immunoblotting. Arrows indicate full-length and processing products of caspases

Article Snippet: Antibodies used for immunoblotting were as follows: anti-human FADD (BD Pharmingen, North Ryde, NSW, Australia; 556402), anti-human FLIP (Enzo Life Sciences, Redfern, NSW, Australia; ALX-804-961-0100), anti-full-length mouse caspase-8 (Enzo Life Sciences; ALX-804-448-C100), anti-cleaved mouse caspase-8 (Cell Signalling Technology, Danvers, MA, USA; 8592), anti-cIAP1 and anti-cIAP2 (Alexis Biochemicals, San Diego, CA, USA; ALX-803-341), anti-XIAP (MBL, M044-3), anti-FLAG M2 (Sigma; F-3165), anti-β-actin (Sigma; A-1978), anti-mouse caspase-8 (Cell Signalling Technology; 4927), anti-human caspase-8 (MBL, Woburn, MA, USA; M058-3), anti-caspase-10 (MBL; M059-3), anti-cleaved caspase-3 (Cell Signalling Technology; 9661), anti-human PKR (Santa Cruz; sc6282), anti-RIPK1 (BD Transduction Laboratories, North Ryde, NSW, Australia, 610458), anti-mouse RIPK3 (Axxora, Farmingdale, NY, USA; PSC-2283-c100), anti-human phospho-MLKL (Abcam, Milton, Cambridge, UK; ab187091), anti-total mouse and human MLKL (housemade, 3H1).

Techniques: CRISPR, Flow Cytometry, Lysis, SDS Page, Western Blot

Journal: Cell Death and Differentiation

Article Title: Combination of IAP antagonist and IFN γ activates novel caspase-10- and RIPK1-dependent cell death pathways

doi: 10.1038/cdd.2016.147

Figure Lengend Snippet: Caspase-10 mediates IFNγ/SM cell death in the absence of caspase-8 and necroptosis. (a) Immunoblot of wild-type and CRISPR/Cas9 MLKL−/−CASP8−/−, RIPK3−/−CASP8−/− HT29 cells treated with IFNγ/SM and IDN-6556 (10 μM) and lysed in SDS lysis buffer. Immunostaining was performed for the indicated proteins. (b) Wild-type (black bar) and CRISPR/Cas9 CASP10−/− (grey bar), RIPK3−/−CASP10−/− (light blue bar), MLKL−/−CASP10−/− (light green bar), RIPK3−/−CASP8−/−CASP10−/− (light blue frame), MLKL−/−CASP8−/−CASP10−/− (light green frame) HT29 cells were treated with IFNγ/SM, IDN-6556 and Nec-1 or not treated (UT) for 48 h before measuring dead cells by detecting PI-permeable cells using flow cytometry. Data are plotted as mean±S.E.M. (n≥3). (c) Wild-type and CRISPR/Cas9 CASP10−/−, MLKL−/−CASP10−/−, MLKL−/−CASP8−/−CASP10−/−, RIPK3−/−CASP8−/−CASP10−/−, RIPK3−/−CASP8−/− HT29 cells were treated as indicated for 24 h, lysed in SDS lysis buffer and immunoblotted. Arrows indicate full-length and processing products of caspases

Article Snippet: Antibodies used for immunoblotting were as follows: anti-human FADD (BD Pharmingen, North Ryde, NSW, Australia; 556402), anti-human FLIP (Enzo Life Sciences, Redfern, NSW, Australia; ALX-804-961-0100), anti-full-length mouse caspase-8 (Enzo Life Sciences; ALX-804-448-C100), anti-cleaved mouse caspase-8 (Cell Signalling Technology, Danvers, MA, USA; 8592), anti-cIAP1 and anti-cIAP2 (Alexis Biochemicals, San Diego, CA, USA; ALX-803-341), anti-XIAP (MBL, M044-3), anti-FLAG M2 (Sigma; F-3165), anti-β-actin (Sigma; A-1978), anti-mouse caspase-8 (Cell Signalling Technology; 4927), anti-human caspase-8 (MBL, Woburn, MA, USA; M058-3), anti-caspase-10 (MBL; M059-3), anti-cleaved caspase-3 (Cell Signalling Technology; 9661), anti-human PKR (Santa Cruz; sc6282), anti-RIPK1 (BD Transduction Laboratories, North Ryde, NSW, Australia, 610458), anti-mouse RIPK3 (Axxora, Farmingdale, NY, USA; PSC-2283-c100), anti-human phospho-MLKL (Abcam, Milton, Cambridge, UK; ab187091), anti-total mouse and human MLKL (housemade, 3H1).

Techniques: Western Blot, CRISPR, Lysis, Immunostaining, Flow Cytometry

Journal: The Journal of Cell Biology

Article Title: Necroptosis mediators RIPK3 and MLKL suppress intracellular Listeria replication independently of host cell killing

doi: 10.1083/jcb.201810014

Figure Lengend Snippet: RIPK3-MLKL pathway prevents systemic spread of Listeria in mice. (A) Human expression levels by tissue for RIPK3 . Gene-level transcripts per kilobase million (TPM) were downloaded from the GTEx portal and summarized across tissue type. Box and whisker plots are plotted in order of highest median expression to lowest. The bottom border, middle line, and top border of the box represent the first quartile, second quartile (median), and third quartile, respectively, while the lines represent 150% of the interquartile range. Outliers more than 150% of the interquartile range are represented as points. 17 representative tissues are shown (see Fig. S1 A for the full results). (B and C) Protein extracts of the indicated tissues from Ripk3 +/+ (WT, #1–3) and Ripk3 −/− (knockout [KO], #1) mice were analyzed by Western blotting with the indicated antibodies. Coomassie brilliant blue (CBB) staining of the whole membrane (B) and β-actin (C) were used as loading controls. (D and E) Ripk3 −/− (KO) and littermate Ripk3 +/+ or Ripk3 +/− (control) mice were orally infected with Listeria . Immunofluorescence staining of liver-colonized Listeria (D) and CFU of Listeria in the liver (E) at 3 d after infection are shown. Arrowheads indicate Listeria in the liver. Bar, 50 µm (mean ± SEM; 6–9-wk-old; control, n = 9; Ripk3 KO, n = 9; *, P < 0.05). (F) Mlkl −/− (KO) and littermate Mlkl +/+ (control) mice were orally infected with Listeria . CFU of Listeria in the liver at 3 d after infection is shown (mean ± SEM; 6–9-wk-old; control, n = 9; Mlkl KO, n = 4; *, P < 0.05).

Article Snippet: Anti–β-actin (AC15; Sigma-Aldrich), anti- Listeria (ab35132; Abcam), anti-mouse RIPK3 (R4277; Sigma-Aldrich), anti-MLKL (EPR17514; Abcam), anti-phospho-human MLKL (Thr357/Ser358; EPR9514; Abcam), anti-FLAG (M2; Sigma-Aldrich), and anti-GFP (GT859; GeneTex; 9F9.F9; Abcam) were used.

Techniques: Expressing, Whisker Assay, Knock-Out, Western Blot, Staining, Infection, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: Necroptosis mediators RIPK3 and MLKL suppress intracellular Listeria replication independently of host cell killing

doi: 10.1083/jcb.201810014

Figure Lengend Snippet: RIPK3-MLKL pathway suppresses intracellular replication of Listeria. (A and B) Control, FLAG-tagged RIPK3 stably expressing (RIPK3+) HeLa cells (A), and HT-29 cells (B) were infected with Listeria (MOI of 10) and cultured for the indicated periods. Phosphorylation levels of MLKL (pMLKL) and RIPK3 (pRIPK3) were analyzed by Western blotting. Gentamicin was added to the culture to eliminate extracellular Listeria . (C and D) Control and RIPK3+ HeLa cells were infected with Listeria (MOI of 10) and cultured for the indicated periods. The cells were stained with anti- Listeria antibody, phalloidin, and DAPI at the indicated time point (C). Bar, 20 µm. Fold replication of Listeria (relative to the number of bacteria at 2 hpi) was measured (D). (E) Control and RIPK3+ HeLa cells were treated with either vehicle (DMSO) or 1 µM GSK’872 for 30 min. The cells were then infected with Listeria (MOI of 10) and cultured for 24 h with either DMSO or 1 µM GSK’872. Listeria CFU relative to vehicle-treated control is shown. (F and G) Control, RIPK3+ HeLa (F), and HT-29 cells (G) were treated with either vehicle (DMSO) or 1 µM NSA for 30 min. The cells were then infected with Listeria (MOI of 10) and cultured for 24 h with either DMSO or 1 µM NSA. Listeria CFU relative to vehicle-treated control is shown (mean ± SEM; n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: Anti–β-actin (AC15; Sigma-Aldrich), anti- Listeria (ab35132; Abcam), anti-mouse RIPK3 (R4277; Sigma-Aldrich), anti-MLKL (EPR17514; Abcam), anti-phospho-human MLKL (Thr357/Ser358; EPR9514; Abcam), anti-FLAG (M2; Sigma-Aldrich), and anti-GFP (GT859; GeneTex; 9F9.F9; Abcam) were used.

Techniques: Stable Transfection, Expressing, Infection, Cell Culture, Western Blot, Staining

Journal: The Journal of Cell Biology

Article Title: Necroptosis mediators RIPK3 and MLKL suppress intracellular Listeria replication independently of host cell killing

doi: 10.1083/jcb.201810014

Figure Lengend Snippet: Listeria invasion–induced phosphorylation of MLKL does not lead to necroptotic cell death. (A) FLAG-tagged RIPK3 stably expressing (RIPK3+) HeLa cells were either treated with 50 ng/ml TNF, 100 nM SMAC mimetic, 20 µM Q-VD-OPh (TSQ) for the indicated time periods, or infected with Listeria (MOI of 10 and 100) for 24 h. Phosphorylation (top panel) and oligomerization (bottom panel) of MLKL were analyzed by Western blotting. *, Nonspecific bands. (B and C) Control and RIPK3+ HeLa cells were either treated with TSQ for the indicated time periods, or infected with Listeria (MOI of 10 and 100) and cultured for 24 h. Phosphorylation level of MLKL (B, Western blotting), bright-field images of the cells (B, right panels), and cell viability measured by crystal violet staining (C) are shown. Bar, 200 µm. (D) Control and FLAG-RIPK3 stably expressing (RIPK3 + ) HeLa cells were either treated with TSQ or infected with Listeria (MOI of 100) and cultured for the indicated time periods. Floating dead cells were washed out after the culture. Membrane permeability of the remaining adherent cells was measured by incorporation of ethidium homodimer III (mean ± SEM; n = 3; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: Anti–β-actin (AC15; Sigma-Aldrich), anti- Listeria (ab35132; Abcam), anti-mouse RIPK3 (R4277; Sigma-Aldrich), anti-MLKL (EPR17514; Abcam), anti-phospho-human MLKL (Thr357/Ser358; EPR9514; Abcam), anti-FLAG (M2; Sigma-Aldrich), and anti-GFP (GT859; GeneTex; 9F9.F9; Abcam) were used.

Techniques: Stable Transfection, Expressing, Infection, Western Blot, Cell Culture, Staining, Permeability