Supplementary Figure S1. TRAIL-induced necroptosis at acidic phe is dependent on
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1 Online supplementary information Supplementary Figure S1. TRAIL-induced necroptos at acidic phe is dependent on RIPK1 and RIPK3. (a) HT29 cells were tranently transfected with RIPK1, RIPK3, RIPK1/ RIPK3 ( RIPK1/3) or 1 (non targeting RNA used as negative control) for 72 h. Dentometry analys of RIPK1 and RIPK3 expreson carried out from three independent western lot experiments. Relative RIPK1 or RIPK3 expreson was expressed in aritrary units (AU) as percentage of asorance measured in 1 transfected cells. () Dentometry analys of RIPK1 and RIPK3 expreson carried out in RIPK1, RIPK1 KO, RIPK3 and RIPK3 KO cells (three independent western lot experiments). Relative RIPK1 or RIPK3 expreson was expressed in aritrary units (AU) as percentage of asorance measured in RIPK1. Mean ± SD. P <.5, P <.1 and P <.1 Supplementary Figure S2. TRAIL-induced necroptos at acidic phe is dependent on TRAIL death receptors. (a) HT29 cells were treated with indicated concentrations of TRAIL-Flag, FasL-Flag (cross-linked with 2 µg/ml anti-flag M2) or TNF, for 24 h, at phyological phe 7.4 or acidic phe 6.5. Percentage of cell death was estimated with a methylene lue viaility assay as descried in M&M. () HT29 cells were treated or not () with 1 ng/ml of TRAIL-Flag (cross-linked with 2 µg/ml anti-flag M2) for 24 h, after a 1 h pre-treatment with 1 µg/ml Etanercept (α TNF) or antagonistic antiodies directed against Fas (α Fas), DR4 (α DR4) or DR5 (α DR5), at phe 7.4 or 6.5. Percentages of apoptos and necros were estimated as descried in M&M. (c) Jurkat cells were treated or not () with 1 ng/ml FasL-Flag (cross-linked with 2 µg/ml anti-flag M2) for 24 h, after a 1 h pretreatment with 1 µg/ml antagonistic antiody directed against Fas (α Fas). Percentage of cell death was estimated as descried in M&M. (d) L929 cells were treated or not () with 1
2 1 ng/ml TNF and µm z-vad for 24 h, after a 1 h pre-treatment with 1 µg/ml Etanercept (α TNF). Percentage of cell death was estimated with a methylene lue viaility assay. Mean ± SD. # P <.5 and $$$, P <.1 Supplementary Figure S3. TRAIL-induced necroptos at acidic phe is dependent on TRAIL death receptors. (a) HT29 cells were tranently transfected with RNAs directed against TNFR1, Fas, DR4 and DR5 or with 1 (non targeting RNA used as negative control). 72 h after transfection, cells were treated or not () with 1 ng/ml TRAIL-Flag and 2 µg/ml anti-flag M2 for 24 h, at phyological phe 7.4 or acidic phe 6.5. Percentages of apoptos and necros were estimated as descried in M&M. () Western lot analys of TNFR1, Fas, DR4 or DR5 expreson in HT29 cells was carried out 72 h after transfection (one representative of three independent experiments). Anti-human HSC7 was used as a control of protein loading. (c) Dentometry analys of TNFR1, Fas, DR4 or DR5 expreson was carried out in HT29 cells 72 h after transfection (three experiments). Relative TNFR1, Fas, DR4 or DR5 expreson was expressed in aritrary units (AU) as percentage of asorance measured in 1 transfected cells., # P <.5, ## P <.1 and, P <.1 Supplementary Figure S4. TRAIL-induced necroptos at acidic phe is dependent on TRAIL death receptors. (a) Jurkat cells were tranently transfected with a RNA directed against Fas or with 1 (non targeting RNA used as negative control). 72 h after transfection, cells were treated or not () with 1 ng/ml FasL-Flag and 2 µg/ml anti-flag M2 for 24 h. Percentages of apoptos and necros were estimated as descried in M&M. () L929 cells were tranently transfected with a RNA directed against TNFR1 or with h after transfection, cells were treated or not () with 1 ng/ml TNF and µm z- VAD for 24 h. Percentage of cell death was estimated with a methylene lue viaility assay as 2
3 descried in M&M. (c) Jurkat cells were tranently transfected with Fas or with 1 for 72 h. Western lot analys of Fas was carried in transfected cells. Anti-human HSC7 was used as a control of protein loading (one representative of three independent experiments). Dentometry analys of Fas expreson was carried out in Jurkat cells 72 h after transfection (three experiments). Relative Fas expreson was expressed in aritrary units (AU) as percentage of asorance measured in 1 transfected cells. (d) L929 cells were tranently transfected with TNFR1 or with 1 for 72 h. Western lot analys of TNFR1 was carried in transfected cells. Anti-human HSC7 was used as a control of protein loading (one representative of three independent experiments). Dentometry analys of TNFR1 expreson was carried out in L929 cells 72 h after transfection (three experiments). Relative TNFR1 expreson was expressed in aritrary units (AU) as percentage of asorance measured in 1 transfected cells. (e) HT29 cells were treated or not () with 1 ng/ml TRAIL-Flag and 2 µg/ml anti-flag M2 for indicated times, at phyological phe 7.4 or acidic phe 6.5. TNF extracellular concentration was determined y ELISA as descried in M&M. Mean ± SD. P <.1 and P <.1 Supplementary Figure S5. TRAIL-induced necroptos at acidic phe is dependent on PARP-1. (a) HT29 cells were treated with 1 ng/ml TRAIL-Flag and 2 µg/ml anti-flag M2, for the indicated times, at phyological phe 7.4 or acidic phe 6.5. Dentometry analys of PAR expreson was carried out from three independent western lot experiments. Relative PAR expreson was expressed in aritrary units (AU) as percentage of asorance measured in cells at time. () HT29 cells were tranently transfected with a RNA directed against PARP-1 or with 1 for 72 h. Dentometry analys of PARP-1 expreson was carried from three independent western lot experiments. Relative PARP-1 expreson was expressed in aritrary units (AU) as percentage of asorance measured in 1 transfected cells. (c) 3
4 MEF PARP-1 or MEFS PARP-1 KO cells were tranently transfected with PARP-1 or 1 for 72 h. Dentometry analys of PARP-1 expreson was carried out from three independent western lot experiments. Relative PARP-1 expreson was expressed in aritrary units (AU) as percentage of asorance measured in 1 transfected cells. Mean ± SD. P <.5, P <.1 and P <.1 Supplementary Figure S6. ROS production in TRAIL-induced necroptos at acidic phe. (a) HT29 cells were treated or not () with 1 ng/ml TRAIL-Flag and 2 µg/ml anti- Flag M2 for the indicated times, at acidic phe 6.5. ROS production was measured y flow cytometry as descried in M&M. Menadione was used as potive control. Left panel: flow cytometry kinetic analys of superoxide anion (O.- 2 ) generation with DHE staining (one representative of three independent experiments is shown); right panel: quantitative analyses of superoxide anion generation measured in three independent experiments. Values are represented as percentage of cells with accumulated O.- 2. () HT29 cells were treated or not () with 1 ng/ml TRAIL-Flag and 2 µg/ml anti-flag M2 for 24 h, at phe 6.5, after a 4 h pretreatment with mm N-acetyl-cysteine (NAC), 1 µm Thiourea or 5 mm TEMPOL. ROS production was measured y flow cytometry as descried in M&M. Quantitative analyses of superoxide anion generation measured in three independent experiments. Values are represented as percentage of cells with accumulated O.- 2. (c) HT29 cells were treated or not () with 1 µm Menadione for 1 h, after a 4 h pretreatment with mm NAC, 1 µm Thiourea or 5 mm TEMPOL. ROS production was measured y flow cytometry as descried in M&M. Quantitative analyses of superoxide anion generation measured in three independent experiments. Values are represented as percentage of cells with accumulated O 2 -. Mean ± SD., # P <.5, ## P <.1 4
5 Supplementary Figure S7. ROS production is not involved in TRAIL-induced necroptos at acidic phe. (a) HT29 cells were treated or not () with 1 ng/ml TRAIL- Flag and 2 µg/ml anti-flag M2 for 24 h, at phyological phe 7.4 or acidic phe 6.5, after a 4 h pretreatment with mm NAC, 1 µm Thiourea or 5 mm TEMPOL. Percentages of apoptos and necros were estimated as descried in M&M. () PARP-1 activity was determined as descried in M&M. (c) HT29 cells were treated or not () with 1 ng /ml TRAIL-Flag and 2 µg/ml anti-flag M2 for 8 h, at phe 7.4 or 6.5, after a 4 h pretreatment with mm NAC, 1 µm Thiourea or 5 mm TEMPOL. Intracellular ATP concentration was measured as descried in M&M. (d) HT29 cells were treated or not () with 15 mm propionate and 3 mm acetate for 24 h, after a 4 h pretreatment with mm NAC or 5 mm TEMPOL. Percentage of cell death was estimated with a methylene lue viaility assay as descried in M&M. (e) ROS production was measured y flow cytometry as descried in M&M. Quantitative analyses of superoxide anion generation measured in three independent experiments. Values are represented as percentage of cells with accumulated O.- 2. Mean ± SD. # P <.5, P <.1 Supplementary Figure S8. Concanavalin A-induced hepatitis is associated with TRAILinduced necrotic cell death. (a) C57Bl/6 mice were treated or not (=PBS, 4 mice) with mg/kg Con A for 6 h (five mice) or 1 h (four mice). Western lot analys of HMGB1 or Cyclophilin A was carried out in lysates of liver tissues. Anti-human HSC7 was used as a control of protein loading. Dentometry analys of HMGB1 or Cyclophilin A (CypA) expreson was carried out. Mean relative HMGB1 or CypA expreson was expressed in aritrary units (AU) as percentage of asorance measured in. () C57Bl/6 mice were treated or not (PBS, five mice) with 12 mg/kg Con A for 1 h (eight mice). Western lot analys of protein poly ADP-riosylation (PAR), PARP-1 and caspase-3 was carried out in 5
6 lysates of liver tissues. Anti-human HSC7 was used as a control of protein loading. Dentometry analys of PAR, PARP-1 or caspase-3 expreson was carried out. Mean relative PAR, PARP-1 or caspase-3 expreson was expressed in aritrary units (AU) as percentage of asorance measured in PBS. Mean ± SD. P <.5, P <.1 and P <.1 6
7 Supplementary Figure S1 a 1,25 1,25 Relative RIPK1 1,,75,5,25 Relative RIPK3 1,,75,5,25,, Relative RIPK1 expreson (AU U) 1,,75,5,25, RIPK1 Relative RIPK3 expreson (AU U) 1,,25 RIPK1 KO RIPK3 RIPK3 KO,75,5, RIPK1 RIPK1 KO RIPK3 RIPK3 KO
8 Supplementary Figure S2 a % cell death HT TNF FasL TRAIL phe 7.4 phe (ng/ml) % cell death $$$ 5 1 (ng/ml) $$$ $$$ % apoptos HT α TNF α Fas 8 6 α DR4 6 4 α DR5 4 TRAIL TRAIL phe 7.4 phe 6.5 % necros # TRAIL TRAIL phe 7.4 phe 6.5 c Jurkat 1 d L929 1 % cell l death ll death % cel α Fas α Fas α TNF α TNF FasL TNF + z-vad
9 Supplementary Figure S3 a % apoptos HT TNFR1 Fas DR4 DR5 ## # % necros ## TRAIL TRAIL TRAIL TRAIL phe 7.4 phe 6.5 phe 7.4 phe TNFR1 Fas DR4 DR5 55 kda 48 kda 5 kda 48 kda TNFR1 Fas DR4 DR5 7 kda HSC7 c 1, 1, Relative TNFR1,75,5,25 Relative Fas,75,5,25,, Relative DR4 1,,75,5,25, Relative DR5 1,,75,5,25,
10 Supplementary Figure S4 a Jurkat 1 L929 1 % cell death % cell death Fas 1 Fas 1 TNFR1 1 TNFR1 FasL TNF + z-vad c 1 Fas d 1 TNFR1 48 kda Fas 55 kda TNFR1 7 kda HSC7 7 kda HSC7 1, 1, Relative Fas,75,5,25, 1 Fas Relative TNFR1,75,5,25, 1 TNFR1 e TNF concentration (pg/ml) HT phe 7.4 phe Time (h)
11 Supplementary Figure S5 a Relative PAR 3, 2,5 2, 1,5 1,,5 phe 7.4 phe 6.5, Time (h) c 1, 1, Relative PARP-1,75,5,25, 1 PARP-1 Relative PARP-1,75,5,25, 1 PARP-1 1 PARP-1 PARP-1 KO PARP-1
12 Supplementary Figure S6 a TRAIL phe 6.5 Numer of cells Menadione 24 h 16 h 12 h 8 h 4 h 2 h TRAIL Percentage of cells with accumulated O DHE fluorescence (O 2.- ) TRAIL phe 6.5 c Percentage of cells with accumulated O NAC Thiourea TEMPOL # # # TRAIL phe 6.5 Percentage of cells with accumulated O NAC Thiourea TEMPOL ## # ## Menadione
13 Supplementary Figure S7 a 1 1 % apoptos NAC Thiourea TEMPOL % necros TRAIL TRAIL TRAIL TRAIL phe 7.4 phe 6.5 phe 7.4 phe 6.5 PARP activity (AU) phe 7.4 phe 6.5 c Intracellular ATP (%//Cell numer) NAC Thiourea TEMPOL TRAIL TRAIL phe 7.4 phe 6.5 TRAIL d e % cell death NAC TEMPOL Propionate / Acetate Percentage of cells with accumulated O NAC TEMPOL # # Propionate / Acetate
14 Supplementary Figure S8 a ConA 6h ConA 1h 25 kda HMGB1 18 kda Cyclophilin A 7 kda HSC7 Mea an Relative CypA ex xpreson (AU) 2, 1,5 1,,5, 6h 1h Mean Relative HM MGB1 expreson (AU) 2, 1,5 1,,5, 6h 1h PBS Con A 1h 116- kda PAR 116 kda PARP-1 32 kda Caspase-3 7 kda HSC7 Mean Relative PARP-1 expreson (AU) 3, 2,5 2, 1,5 1,,5, 3, PBS 1h Mean Relative PA AR 3, 2,5 2, 1,5 1,,5, PBS 1h Mean Relative Caspase-3 2,5 2, 1,5 1,,5, PBS 1h
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Figure S1. Western blot analysis of clathrin RNA interference in human DCs Human immature DCs were transfected with 100 nm Clathrin SMARTpool or
Figure S1. Western blot analysis of clathrin RNA interference in human DCs Human immature DCs were transfected with 100 nm Clathrin SMARTpool or control nontargeting sirnas. At 90 hr after transfection,
Supplementary Figure 1. Deletion of Smad3 prevents B16F10 melanoma invasion and metastasis in a mouse s.c. tumor model.
A B16F1 s.c. Lung LN Distant lymph nodes Colon B B16F1 s.c. Supplementary Figure 1. Deletion of Smad3 prevents B16F1 melanoma invasion and metastasis in a mouse s.c. tumor model. Highly invasive growth
Supplementary Figure 1
Supplementary Figure 1 Supplementary Figure 1 Schematic depiction of the tandem Fc GDF15. Supplementary Figure 2 Supplementary Figure 2 Gfral mrna levels in the brains of both wild-type and knockout Gfral
Supplementary Figure 1 Maschalidi et al.
a 1% 5% % 1% 5% % OVAb βgal activity A.U. (x1 4 ) 2 1 5 βgal activity A.U. (x1 4 ) 2 1 BSAb 2 hours 4 hours OVAb BSAb OVAb BSAb,1 1 1 1 SIINFEKL (ng/ml) CFSE b Beads Alexa488 (%) 8 6 4 2 ** ** 1:1 5:1
Supplemental Figure 1.
antifas CD40L 0 15 30 60 0 15 30 60 min piκbα IκBα p38 Supplemental Figure 1. CD40L and antifas stimulation induces NFkB activation. BMDM were stimulated with CD40L (1ug/ml) or antifas (10ug/ml). Cell
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Tbk1-TKO! DN cells (%)! 15! 10!
a! T Cells! TKO! B Cells! TKO! b! CD4! 8.9 85.2 3.4 2.88 CD8! Tbk1-TKO! 1.1 84.8 2.51 2.54 c! DN cells (%)! 4 3 2 1 DP cells (%)! 9 8 7 6 CD4 + SP cells (%)! 5 4 3 2 1 5 TKO! TKO! TKO! TKO! 15 1 5 CD8
Supplementary information
Supplementary information Supplementary Figure S1: Ex[Ca 2+ ]-induced IL-1ß production of monocytes primed with different TLR ligands IL-1ß release of CD14+ monocytes in response to stimulation for 16
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Supplementary Information File
Supplementary Information File Supplementary Table 1. List of synthesized sirna sequences for target genes sirna Species Sequence Ctrl sirna mouse sense 5 -UUCUCCGAACGUGUCACGUTT-3 Antisense 5 -ACGUGACACGUUCGGAGAATT-3
Supplementary Fig. 1 No relative growth advantage of Foxp3 negative cells.
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Supplementary Figure 1 YAP negatively regulates IFN- signaling. (a) Immunoblot analysis of Yap knockdown efficiency with sh-yap (#1 to #4 independent constructs) in Raw264.7 cells. (b) IFN- -Luc and PRDs
Supplementary Figure 1
Supplementary Figure 1 Expression of apoptosis-related genes in tumor T reg cells. (a) Identification of FOXP3 T reg cells by FACS. CD45 + cells were gated as enriched lymphoid cell populations with low-granularity.
well for 2 h at rt. Each dot represents an individual mouse and bar is the mean ±
Supplementary data: Control DC Blimp-1 ko DC 8 6 4 2-2 IL-1β p=.5 medium 8 6 4 2 IL-2 Medium p=.16 8 6 4 2 IL-6 medium p=.3 5 4 3 2 1-1 medium IL-1 n.s. 25 2 15 1 5 IL-12(p7) p=.15 5 IFNγ p=.65 4 3 2 1
Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk
Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk -/- mice were stained for expression of CD4 and CD8.
Nature Immunology: doi: /ni.3866
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DOI: 10.1038/ncb3461 In the format provided by the authors and unedited. Supplementary Figure 1 (associated to Figure 1). Cpeb4 gene-targeted mice develop liver steatosis. a, Immunoblot displaying CPEB4
Tumor suppressor Spred2 interaction with LC3 promotes autophagosome maturation and induces autophagy-dependent cell death
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Supplementary Figure 1. Behavioural effects of ketamine in non-stressed and stressed mice. Naive C57BL/6 adult male mice (n=10/group) were given a single dose of saline vehicle or ketamine (3.0 mg/kg,
Introduction: 年 Fas signal-mediated apoptosis. PI3K/Akt
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SUPPLEMENTARY FIGURES Supplementary Figure 1: immunoprecipitation with anti-casr antibody The Casr protein was expressed in transiently transfected HEK cells. Cell lysates from HEK cells were subjected
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DOI:.38/ncb3399 a b c d FSP DAPI 5mm mm 5mm 5mm e Correspond to melanoma in-situ Figure a DCT FSP- f MITF mm mm MlanaA melanoma in-situ DCT 5mm FSP- mm mm mm mm mm g melanoma in-situ MITF MlanaA mm mm
Time after injection (hours) ns ns
Platelet life span (% iotinylated platelets) 1 8 6 4 2 4 24 48 72 96 Time after injection (hours) 6 4 2 IgG GPIα GPIβ GPII GPVI Receptor expression (GeoMean, fluorescence inteity) Supplementary figure
Supplementary Material
Supplementary Material Summary: The supplementary information includes 1 table (Table S1) and 4 figures (Figure S1 to S4). Supplementary Figure Legends Figure S1 RTL-bearing nude mouse model. (A) Tumor
genome edited transient transfection, CMV promoter
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Supplementary Figure 1. Dynamic Response of WT and mir-21 -/- mice to caerulein. (a) Representative histological sections of mouse pancreas stained
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Supplemental Figure 1
Supplemental Figure 1 1a 1c PD-1 MFI fold change 6 5 4 3 2 1 IL-1α IL-2 IL-4 IL-6 IL-1 IL-12 IL-13 IL-15 IL-17 IL-18 IL-21 IL-23 IFN-α Mut Human PD-1 promoter SBE-D 5 -GTCTG- -1.2kb SBE-P -CAGAC- -1.kb
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Supplementary Material HLA-DM Captures Partially Empty HLA-DR Molecules for Catalyzed Peptide Removal Anne-Kathrin Anders, Melissa J. Call, Monika-Sarah E. D. Schulze, Kevin D. Fowler, David A. Schuert,
Supplementary Figure 1 Chemokine and chemokine receptor expression during muscle regeneration (a) Analysis of CR3CR1 mrna expression by real time-pcr
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Supplementary Figure 1
Combination index (CI) Supplementary Figure 1 2. 1.5 1. Ishikawa AN3CA Nou-1 Hec-18.5...2.4.6.8 1. Fraction affected (Fa) Supplementary Figure 1. The synergistic effect of PARP inhibitor and PI3K inhibitor
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of whole cell cultures in U-bottomed wells of a 96-well plate are shown. 2
Supplementary online material Supplementary figure legends Supplementary Figure 1 Exposure to T reg cells causes loss of T resp cells in co-cultures. T resp cells were stimulated with CD3+CD28 alone or
Supplementary Materials
Supplementary Materials Figure S1. MTT Cell viability assay. To measure the cytotoxic potential of the oxidative treatment, the MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide] assay
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doi:10.1038/nature11700 Figure 1: RIP3 as a potential Sirt2 interacting protein. Transfected Flag-tagged Sirt2 was immunoprecipitated from cells and eluted from the Sepharose beads using Flag peptide.