Supplementary Figure 1. AnnexinV FITC and Sytox orange staining in wild type, Nlrp3 /, ASC / and casp1/11 / TEC treated with TNF /CHX.
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1 Supplementary Figure 1. AnnexinV FITC and Sytox orange staining in wild type, Nlrp3 /, ASC / and casp1/11 / TEC treated with TNF /CHX. Phase contrast and widefield fluorescence microscopy (20x magnification). AnnexinV staining is reduced in Nlrp3 / and ASC / cells but not casp1/11 / TEC.
2 Supplementary Figure 2. A. MTT cell death assay in wild type and Nlrp3 / TEC activated with anti CD95 antibody at 6, 12 and 24 hours. B, C. Quantification of Annexin V FITC and Sytox orange fluorescence in wild type and Nlrp3 / TEC treated with anti CD95. Wild type vs Nlrp3 /, *p<0.05 n=3 independent experiments. These data show that Nlrp3 regulates CD95 mediated apoptosis. D. Immunoblotting for caspase 8 p18 fragment in wild type or Nlrp3 / TEC treated with TNF /CHX or anti CD95 over a 48 hour time course.
3 Supplementary Figure 3. A. Immunoblotting for indicated proteins demonstrating similar levels of expression in wild type and Nlrp3 / TEC untreated or stimulated with TNF /CHX at 24 hours. B. Quantification of AnnexinV staining in wild type or Nlrp3 / TEC stimulated with the indicated conditions at 24 hours (mean +/ s.e.m., * p<0.05, **p<0.01, n=3 independent experiments). The SMAC mimetic birinapant enhances both TNF and CD95 induced apoptosis.
4 Supplementary Figure 4. A. Immunblotting for cleaved caspase 8 (p43 and p18) in primary mouse peritoneal macrophage stimulated with TNF /CHX for the indicated time points. Nlrp3 is dispensable for TNF /CHX induced caspase 8 activation in macrophages. B. Quantification of AnnexinV staining in wild type or Nlrp3 / TEC stimulated with TNF or anti CD95andbothCHX and the SMAC mimetic birinapant at 24 hours (mean +/ s.e.m., p=ns, n=3 independent experiments). Nlrp3 is dispensable for death receptor mediated apoptosis in birinapant and CHXtreated TEC.
5 Supplementary Figure 5. Canonical inflammasome activation in bone marrow derived macrophages. Wild type, Nlrp3 / and Casp1/11 / BMDMs were treated with 20 μm nigericin for one and three hours. Cell extracts and supernatants were analyzed by immunoblotting for cleaved caspase 1 (p10) and cleaved caspase 8. Nigiricin induces rapid and efficient cleavage and secretion of caspase 1 and caspase 8 in wild type cells, and only caspase 8 in casp1/11 / macrophages.
6 Supplementary Figure 6. A. Caspase 1 expression (immunoblotting) in mouse TEC treated with TNF (1, 10 ng/ml) or LPS (10, 100 ng/ml) at 24 hours. Bone marrow derived macrophages and Casp1/11 / are shown as positive and negative controls. B. Nlrp3 expression at 24 hours in mouse TEC stimulated with TNF (1, 10 ng/ml) and LPS (10, 100 ng/ml). C. Role of Caspase 11 in TEC apoptosis. Immunoblotting for caspase 11 in wild type, Nlrp3 / and casp1/11 / TECs after 24 and 48 hours of TNFα (10 ng/ml) and cycloheximide (5 µg/ml) treatment. Caspase 11 p10 or p20 cleavage products are not induced during TNFmediated cell death. D. IL 18ELISAinBMDM(6hours)orTECtreatedwithATPorTNF /CHX at 24 hours. All cells were primed with 100 ng/ml of LPS. E. NLRP3 and caspase 1 expression in HPTC treated with increasing concentrations of TNF (0.1,1,10ng/ml)orLPS(1,10and 100 ng/ml) at 24 hours. Priming has little to no effect on caspase 1, NLRP3 expression or canonical inflammasome activation in tubular epithelial cells
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8 Supplementary Figure 7. A. Confocal immunofluorescence microscopy probing for NLRP3 and ASC in HPTC untreated and following stimulation with TNF /CHX at 24 hours (X40 lens X2 zoom). B. Mouse and rabbit isotype controls for NLRP3 (mouse) and ASC (rabbit) antibodies in untreated and TNF /CHX treated cells at 24 hours (X40 lens X2 zoom). C. Confocal immunofluorescence microscopy probing for NLRP3 and the Golgi marker GM130. Untreated and TNF /CHX treated HPTC at 24 hours (X60 lens X3 zoom). NLRP3 does not localize to Golgi. D. Immunofluorescence confocal microscopy of HPTC treated with TNF /CHX at 24 hours probing for NLRP3 and the lysosomal marker LAMP1. LAMP1 does not co localize to NLRP3 complexes during apoptosis. E. ASC specks in epithelial cells. Confocal immunofluorescence microscopy in control and TNF /CHX treated HPTC probing for ASC (red) and E cadherin (green). ASC specks in E cadherin positive cells confirm epithelial cells. All cells were background stained with DAPI (blue) (X40 lens X2 zoom).
9 Supplementary Figure 8. Caspase 1 activation in HPTC and THP 1 cells. A. Confocal immunofluorescence microscopy in control and TNF /CHX treated HPTC probing for activatedcaspase 1(red)and caspase 8 (green). Caspase 8 but not caspase 1 is activated during apoptosis in HPTC. B. Confocal immunofluorescence microscopy in control and nigericin treated THP 1 macrophages probing for activated caspase 1 (red)and ASC (green). Images confirm canonical inflammasome activation and integrity of the caspase 1 FLICA probe. All cells were background stained with DAPI (blue) (X60 lens X3 zoom).
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11 Supplementary Figure 9. A. Confocal immunofluorescence microscopy in HTPC treated with activating anti CD95 antibody at 24 hours. Probing for caspase 8 (FLICA, green), NLRP3(red) and ASC (magenta) (X60 lens X3 zoom). NLRP3, ASC and activated caspase 8 colocalize downstream of CD95. B. ASC specks in wild type and ASC / mousetectreatedwithtnf /CHX at 24 hours. Activated caspase 8 (FLICA, green) co localizes to ASC specks in TEC following 24 hours of TNF /CHX treatment. Rabbit isotype controls are negative. ASC / TEC do not stain for ASC as expected, although some active caspase 8 can be detected (top panels X40 lens X2 zoom, bottom panels X60 lens X3 zoom). These data are consistent with the biochemical studies that show incomplete impairment of caspase 8 activation in ASC / cells, likely due to ongoing death receptor signaling and possibly redundant pathways.
12 Supplementary Figure 10. Caspase 8 activation in THP 1 cells stimulated with TNF /CHX at 6 hours. A. Staining for activated caspase 8 (FLICA, green), NLRP3 (red) and ASC (magenta). B. Staining for caspase 8 and Mitotracker Red (X60 lens X3 zoom). Unlike epithelial cells, caspase 8 activation downstream of TNFR is not localized to NLRP3, ASC or mitochondria. Furthermore, NLRP3/ASC specks are not observed.
13 Supplementary Figure 11. Caspase 8 is not activated by the canonical NLRP3 agonist nigericin in HPTC. Confocal immunofluorescence microscopy probing for activated caspase 8 (FLICA, green), NLRP3 (red) and ASC (magenta) in LPS primed and unprimed HPTC treated with nigericin (20 M) for 24 hours (X60 lens X3 zoom).
14 Supplementary Figure 12. NLRP3 expression following apoptosis. HPTC were treated with TNF /CHX for 24 hours and NLRP3 was immunoprecipitated from protein lysates followed by immunoblotting. NLRP3 forms large oligomers (arrow) that are not detectable in extracts following TNF /CHX induced apoptosis.
15 Supplementary Figure 13. Effect of the RIP1 inhibitor necrostatin 1 on epithelial cell apoptosis. A. Immunoblot probing for caspase 8 intnf /birinapant treated mouse TEC (wild type). B. Immunoblotting using antibodies specific for procaspase 8, caspase 8 p43 and capsase 8 p43/p18 in mouse TEC treated with TNF /CHX. Necrostatin inhibits TNF /birinapant but not TNF /CHX induced apoptosis. C. Immunofluorescence microscopy probing for activated caspase 8 (FLICA, green), NLRP3 (red) and ASC (magenta) in HPTC treated with TNF /CHX and necrostatin (X60 lens X3 zoom). TNF /CHX induced NLRP3/ASC/caspase 8 complex formation is not dependent on RIP1
16 Supplementary Figure 14. Nlrp3 regulates caspase 8 activation via non death receptor induced apoptosis. A. Immunoblotting for procaspase 8 and cleaved caspase 8 (p43/p18) in wild type mousetectreatedwithchxalone(5 g/ml), TNF alone (10 ng/ml) or both at 24 hours. B. Quantification of cell death by MTT assay. C. Quantification of AnnexinV FITC labeling (fluorescence). CHX alone induces a small amount of apoptosis and caspase 8 cleavage that is dependent on Nlrp3. D. Caspase 8 activation (immunoblotting) in wild type and Nlrp3 / TEC treated with increasing concentration of etoposide (250, 500, 1000 M) at 6 hours. Nlrp3 also regulates intrinsic activated caspase 8. E. Caspase 9 activation (immunoblotting) in wild type and Nlrp3 / TEC treated with increasing concentration of etoposide ( M) at 6 hours. Nlrp3 also regulatesnon deathreceptor mediated or intrinsic apoptosis.
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