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1 Deegan S, Saveljeva S, Logue SE, Pakos-Zebrucka K, Gupta S, Vandenabeele P, Bertrand MJ,Samali A. (2014) Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions. Autophagy. 2014;10(11): doi: / Figure S1. Analysis of the role of mitochondria in cells compromised in the intrinsic cell death pathway. (A) Casp9 +/+ and casp9 -/- MEFs were treated with 0.5 μm of Tg and 1 μg/ml of Tm for 24 h. CCCP was added for 6 h. Mitochondrial potential was analyzed by evaluating the amount of TMRE positive cells by flow cytometry. (B) Bax +/+ Bak1 +/+ and bax -/- bak1 -/- MEFs were treated with 0.5 μm of Tg for 48 and 72 h and harvested lysates were analyzed for the expression of cleaved CASP8. Actin was used as a loading control. (C) Casp9 +/+ were treated for with 0.5 μg/ml of Tm alone or in combination with Boc-D-FMK. casp9 -/- MEFs were treated for 72 h with 0.5 μg/ml of Tm. After indicated timepoints treatment was removed and colonies were left to form for 10 d. Boc-D-FMK was replenished for the first 3 d after the removal of the treatment. Colonies were stained with crystal violet. Results are representative of at least 3 independent experiments. Error bars represent the mean ± SD. 1
2 Figure S2. Validation of the blocking antibodies for the death receptor-induced cell death. (A) casp9 -/- MEFs were pretreated with 1 μg/ml of cycloheximide and 50 ng/ml of TNF blocking antibody, 100 ng/ml of FAS:Fc or 500 ng/ml of TNFSF10 blocking antibody in case of samples cotreated with blocking antibodies. One h later treatment with 100 ng/ml of TNF, 50 ng/ml of FAS or 50 ng/ml of SuperKillerTRAIL (TNFSF10) was performed. Cell viability was analyzed by propidium iodide exclusion measured by flow cytometry. (B) Casp9 +/+ MEFs were pretreated with 1 μg/ml of cycloheximide and 500 ng/ml of TNFSF10 blocking antibody for 1 h. 50 ng/ml of SuperKillerTRAIL (TNFSF10) was added for 24 h and cell viability was measured by propidium iodide (PI) staining. Results are representative of at least 3 independent experiments. Error bars represent the mean ± SD. 2
3 Figure S3. Autophagic flux in casp9 -/- MEFs deficient for autophagy genes. (A and B) casp9 -/- MEFs stably expressing pgipz or Atg5 shrna were treated with 20 μm chloroquine alone, 0.5 μg/ml Tm alone, or a combination of 20 μm chloroquine and 0.5 μg/ml of Tm for the indicated times and cell lysates immunoblotted for LC3 and actin. (C and D) casp9 -/- MEFs stably expressing plko or Atg7 shrna were treated with 20 μm chloroquine alone, 0.5 μg/ml Tm alone, or a combination of 20 μm chloroquine and 0.5 μg/ml of Tm for the indicated times and cell lysates immunoblotted for LC3 and actin. Results are representative of at least 3 independent experiments. Error bars represent the mean ± SD. 3
4 Figure S4. Knockdown of autophagy genes in Casp9 +/+ and casp9 -/- MEF cells has different outcomes on ER stress-induced cell death. Casp9 +/+ MEFs stably expressing pgipz or Atg5 shrna were generated and (A) treated with 0.5 μm Tg for 24 h after which RNA was extracted, Atg5 and Gapdh expression analyzed by RT-PCR. (B) Casp9 +/+ pgipz and Atg5 shrna MEFs were treated with 0.5 μg/ml of Tm for indicated times and cell viability analyzed by propidium iodide (PI) uptake. (C) casp9 -/- MEFs stably expressing plko or Atg7 shrna were generated via lentiviral transduction. RNA from plko and Atg7 shrna casp9 -/- MEFs was isolated and cdna expression of Atg7 and Gapdh analyzed by RT-PCR. (D) plko and Atg7 shrna casp9 -/- MEFs were treated for the indicated times with 0.5 μm Tg, cell lysates prepared and immunoblotted for cleaved CASP8, cleaved CASP3, and actin. (E) plko and Atg7 shrna casp9 -/- MEFs were treated for the indicated times with 0.5 μm Tg and cell viability analyzed by propidium iodide (PI) uptake. Results are representative of at least 3 independent experiments. Error bars represent the mean ± SD. 4
5 Figure S5. Inhibition of RIP1 kinase activity has no effect on ER stress-induced cell death in casp9 -/- MEFs. casp9 -/- MEFs were treated for indicated times with 0.5 μm Tg (A) and 0.5 μg/ml of Tm (B) alone or in combination with 20 μm of Necrostatin 1. Cell viability was evaluated by propidium iodide (PI) uptake. Results are representative of 3 independent experiments. Error bars represent the mean ± SD. 5
6 Figure S6. Apoptosome-compromised cells undergo cell death in response to genotoxic stress. Casp9 +/+ and casp9 -/- MEFs were treated for indicated times with 50 μm etoposide. (A) lysates immunoblotted for CASP9, CASP3, PARP and actin. (B) Cell viability was analyzed by PI uptake. (C) casp9 -/- MEFs were treated for indicated times with 50 μm etoposide after which CASP8 processing was assessed by immunoblotting. (D) plko and Casp8 shrna casp9 -/- MEFs were treated with 50 μm etoposide for the indicated times and cell viability analyzed by propidium iodide (PI) uptake. 6
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Supplementary figure legends
Supplementary figure legends Supplementary Figure 1. Exposure of CRT occurs independently from the apoptosisassociated loss of the mitochondrial membrane potential (MMP). (A) HeLa cells treated with MTX
Supporting Information. FADD regulates NF-кB activation and promotes ubiquitination of cflip L to induce. apoptosis
1 2 Supporting Information 3 4 5 FADD regulates NF-кB activation and promotes ubiquitination of cflip L to induce apoptosis 6 7 Kishu Ranjan and Chandramani Pathak* 8 9 Department of Cell Biology, School
Supplementary Figure 1
A B D Relative TAp73 mrna p73 Supplementary Figure 1 25 2 15 1 5 p63 _-tub. MDA-468 HCC1143 HCC38 SUM149 MDA-468 HCC1143 HCC38 SUM149 HCC-1937 MDA-MB-468 ΔNp63_ TAp73_ TAp73β E C Relative ΔNp63 mrna TAp73
Plasma exposure levels from individual mice 4 hours post IP administration at the
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Supplementary fig. 1. Crystals induce necroptosis does not involve caspases, TNF receptor or NLRP3. A. Mouse tubular epithelial cells were pretreated
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SUPPLEMENTARY INFORMATION
Figure S1 Induction of non-apoptotic death of SV40-transformed and primary DKO MEFs, and DKO thymocytes. (A-F) STS-induced non-apoptotic death of DKO MEF. (A, B) Reduced viability of DKO MEFs after exposure
Supporting Information. Epigallocatechin-3-gallate (EGCG) promotes autophagy-dependent survival via
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Table S1. New colony formation 7 days after stimulation with doxo and VCR in JURKAT cells
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Supplementary Materials and Methods
Supplementary Materials and Methods Immunoblotting Immunoblot analysis was performed as described previously (1). Due to high-molecular weight of MUC4 (~ 950 kda) and MUC1 (~ 250 kda) proteins, electrophoresis
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Supplementary figure legends Supplementary Figure 1. Expression of Shh signaling components in a panel of gastric cancer. (A) RT-PCR for components of the Shh/Gli pathway in normal fetus cell (MRC-5) and
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Description of Supplementary Files. File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables
Description of Supplementary Files File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables Supplementary Figure 1: (A), HCT116 IDH1-WT and IDH1-R132H cells were
Supplementary Materials for
www.sciencetranslationalmedicine.org/cgi/content/full/8/339/339ra69/dc1 Supplementary Materials for The caspase-8 inhibitor emricasan combines with the SMAC mimetic birinapant to induce necroptosis and
BIK BIM NOXA PUMA MCL-1. p53
HT116 cells A IK IM NOXA PUMA ML-1 p53 48 24 48 24 48 24 48 24 48 24 48 24 48 24 48 24 48 Procaspase 3 PARP leaved Product 12 8 4 24 hr 48 hr Figure S1. HT116 cell death by different proteasome inhibitors.
(A) Dose response curves of HMLE_shGFP (blue circle), HMLE_shEcad (red square),
Supplementary Figures and Tables Figure S1. Validation of EMT-selective small molecules (A) Dose response curves of HMLE_shGFP (blue circle), HMLE_shEcad (red square), and HMLE_Twist (black diamond) cells
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Supplemental Figures:
Supplemental Figures: Figure 1: Intracellular distribution of VWF by electron microscopy in human endothelial cells. a) Immunogold labeling of LC3 demonstrating an LC3-positive autophagosome (white arrow)
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SUPPLEMENTARY INFORMATION doi:1.138/nature9814 a A SHARPIN FL B SHARPIN ΔNZF C SHARPIN T38L, F39V b His-SHARPIN FL -1xUb -2xUb -4xUb α-his c Linear 4xUb -SHARPIN FL -SHARPIN TF_LV -SHARPINΔNZF -SHARPIN
Supplementary Figures
Supplementary Figures Figure S1. Validation of kinase regulators of ONC201 sensitivity. Validation and screen results for changes in cell viability associated with the combination of ONC201 treatment (1
SUPPLEMENTARY FIGURE LEGENDS. atypical adenomatous hyperplasias (AAH); Grade II: adenomas; Grade III: adenocarcinomas;
SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure S1: Tumor grades in Ras G12D ; p53 / lung tumors. Representative histology (H&E) of K-Ras G12D ; p53 / lung tumors 13 weeks after tumor initiation. Grade
Supporting Information
Copyright WILEY-VCH Verlag GmbH & Co. KGaA, 69469 Weinheim, Germany, 212. Supporting Information for Adv. Funct. Mater., DOI:.2/adfm.2122233 MnO Nanocrystals: A Platform for Integration of MRI and Genuine
Supplementary Figure S1 Supplementary Figure S2
Supplementary Figure S A) The blots shown in Figure B were qualified by using Gel-Pro analyzer software (Rockville, MD, USA). The ratio of LC3II/LC3I to actin was then calculated. The data are represented
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SUPPLEMENTARY INFORMATION
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.
Index 255. B lymphocytes, 26 Bak, 96 basal condition preparation, See also apoptosis measurement
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T H E J O U R N A L O F C E L L B I O L O G Y
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Supporting Information
Supporting Information van der Windt et al. 10.1073/pnas.1221740110 SI Materials and Methods Mice and Reagents. C57BL/6 and major histocompatibility complex class I-restricted OVA-specific T-cell receptor
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Supplementary Data Table of Contents:
Supplementary Data Table of Contents: - Supplementary Methods - Supplementary Figures S1(A-B) - Supplementary Figures S2 (A-B) - Supplementary Figures S3 - Supplementary Figures S4(A-B) - Supplementary
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Supplementary Figure 1
Supplementary Figure 1 ZV 50 nm Relative to Protein Levels () C Relative to Protein Levels () 6 4 2 0 10 8 6 4 2 0 Treatment Time (6 h) ZV Concentration (25 µm) ZV Concentration (25 µm) Supplementary Figure
Supplementary Figure 1: Characterisation of phospho-fgfr-y463 antibody. (A)
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Supplementary Figure 1.
Supplementary Figure 1. Increased β cell mass and islet diameter in βtsc2 -/- mice up to 35 weeks A: Reconstruction of multiple anti-insulin immunofluorescence images showing differences in β cell mass
Supplementary Information
Supplementary Information Supplementary Methods Assessment of clinical characteristics of the single-agent anti-pd-1 cohort Clinical data were retrospectively collected from patient record, including age,
Supplementary Data. Supplementary Methods:
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SHREE ET AL, SUPPLEMENTAL MATERIALS. (A) Workflow for tumor cell line derivation and orthotopic implantation.
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Hopkins University, Howard Hughes Medical Institute, USA) (27). Cells were maintained in DMEM
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Supplementary Figure 1 P53 is degraded following Chlamydia infection independent of the cell lysis and protein sample preparation procedure applied.
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http / / cjbmb. bjmu. edu. cn Chinese Journal of Biochemistry and Molecular Biology COX-2 NTera-2 NTera-2 RT-PCR FasL caspase-8 caspase-3 PARP.
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supplementary information
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Supplemental Table I.
Supplemental Table I Male / Mean ± SEM n Mean ± SEM n Body weight, g 29.2±0.4 17 29.7±0.5 17 Total cholesterol, mg/dl 534.0±30.8 17 561.6±26.1 17 HDL-cholesterol, mg/dl 9.6±0.8 17 10.1±0.7 17 Triglycerides,
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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
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Supplementary Materials and Methods
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Supplementary Figures Supplementary Figure 1 Increased ABHD5 expression in human colon cancer associated macrophages. (a) Murine peritoneal macrophages were treated with regular culture medium (Ctrl) or
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SUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION doi:10.1038/nature11429 S1a 6 7 8 9 Nlrc4 allele S1b Nlrc4 +/+ Nlrc4 +/F Nlrc4 F/F 9 Targeting construct 422 bp 273 bp FRT-neo-gb-PGK-FRT 3x.STOP S1c Nlrc4 +/+ Nlrc4 F/F casp1