Supplemental Data
Yüklə 42,36 Kb.
|
- Bu səhifədəki naviqasiya:
- Table S1.
- Supplementary Figure S2.
- Supplementary Figure S3. Thapsigargin and tunicamycin induce DP5 expression in INS-1E cells
- Supplementary Figure S4. ER stress induces cytochrome c release from the mitochondria in INS-1E cell s
- Supplementary Figure S5. CPA and thapsigargin induce higher expression of ER stress markers than tunicamycin in INS-1E cells
- Supplementary Figure S6. CPA increases DP5 expression in primary rat β-cells and human dispersed islet cells
- Supplementary Figure S7. ER stress induces JNK/c-Jun phosphorylation in INS-1E cell s
- Supplementary Figure S8. JunB modulates DP5 expression
- Supplementary Figure S9. Adenoviral inactivation of NF-κB or L-NMA addition decrease cytokine-mediated NO formation in INS-1E cells
- Supplementary Figure S10. IL-1β + IFN-γ-induced DP5 upregulation precedes Chop activation
- Supplementary References
|
Supplementary information Signaling by IL-1β + IFN-γ and ER stress converge on DP5/Hrk activation: a novel mechanism for pancreatic β-cell apoptosis Esteban N. Gurzov, Fernanda Ortis, Daniel A. Cunha, Geoffrey Gosset, Mingtao Li, Alessandra K. Cardozo and Décio L. Eizirik
Table S1. Primer sequences for real time RT-PCR Supplementary Figure S1. (A) Western blot analysis of cytoplasmic cytochrome c in INS-1E cells exposed for 24 h to cytokines and/or L-NMA. β-actin was used as loading control. (B) Time course of Bcl-2 and Bcl-XL protein expression in INS-1E cells exposed to cytokines. (C) Expression of DP5 determined by real time RT-PCR in INS-1E cells treated with IL-1β, IFN-γ or cytokine combination. Values are represented as fold induction compared to control (non-cytokine treated cells). *P < 0.05, **P < 0.01 vs. control. (D) cDNA was obtained from human islets of three organ donors as previously described.1,2 The human islets were shipped to Brussels for study within 1-5 days of isolation. After overnight recovery in Ham's F-10 medium containing 6.1 mM glucose, 2 mM GlutaMAX, 50 µM 3-isobutyl-1-methylxanthine, 1% BSA, 50 U/ml penicillin, 50 µg/ml streptomycin and 10% FCS, islets were exposed to cytokines (human recombinant IL-1β (50 U/ml) + human recombinant IFN-γ (1000 U/ml, R&D systems)) in the same medium without FCS for 24 or 48 h as described.2 The percentage of β-cells (45-70%) was assessed in dispersed-islet preparations following staining with mouse monoclonal anti-insulin antibody (1:1000, Sigma) and donkey anti-mouse IgG rhodamine (1:200, Jackson ImmunoResearch Europe, Soham, Cambridgeshire, UK).
INS-1E cells were transfected with inactive siRNA (Control) or two different DP5 siRNAs (siRNA DP5-1 and siRNA DP5-2) and treated with IL-1β + IFN-γ for 24 h. Apoptosis was measured using the Cell Death Detection ELISAplus kit (Roche Diagnostics, Vilvoorde, Belgium), which detects cytoplasmic fragmented DNA. Results are expressed as arbitrary units of optical densities (O.D.). *P < 0.05. Supplementary Figure S3. Thapsigargin and tunicamycin induce DP5 expression in INS-1E cells (A-B) Time course analysis of thapsigargin- or tunicamycin- induced DP5 mRNA expression, as analyzed by real time RT-PCR. *P < 0.05, ***P < 0.001 vs. 0 h. (C-D) INS-1E cells were transfected with control or DP5 siRNAs and real-time PCR was performed 4 h and 8 h after addition of the ER stressors. *P < 0.05, **P < 0.01. (E-F) siRNA-mediated DP5 knockdown protected INS-1E cells against cell death induced by a 24 h exposure to thapsigargin or tunicamycin. *P < 0.05, **P < 0.01. Supplementary Figure S4. ER stress induces cytochrome c release from the mitochondria in INS-1E cells Immunofluorescence analysis of cytochrome c (red) and AIF (green) in INS-1E cells treated with different ER stressors. Nuclear morphology is shown by Hoechst-staining (blue). Arrows indicate compaction of the nuclear chromatin and cytochrome c release from the mitochondria, bar 20m. Supplementary Figure S5. CPA and thapsigargin induce higher expression of ER stress markers than tunicamycin in INS-1E cells INS-1E cells were treated with CPA, thapsigargin or tunicamycin for 8 h and real time RT-PCR for XBP-1s and Chop expression was performed. *P < 0.05, ***P < 0.001 vs. control. Supplementary Figure S6. CPA increases DP5 expression in primary rat β-cells and human dispersed islet cells (A) Purified rat β-cells were treated with CPA for 24 h and real time RT-PCR for DP5 was performed. *P < 0.05 vs. control. (B) Human dispersed islet cells (cultured as described in Supplementary Figure 1C) were treated with CPA (50 M) for 24 h and real time RT-PCR for DP5 was performed. *P < 0.05 vs. control. (C) DP5 knockdown decreased CPA-induced apoptosis in rat primary β-cells. Cell death was evaluated by HO/PI 24 h after CPA treatment. *P < 0.05. Supplementary Figure S7. ER stress induces JNK/c-Jun phosphorylation in INS-1E cells After different time points of exposure to ER stressors, Western blot analysis was performed to determine changes in JNK and c-Jun phosphorylation. Supplementary Figure S8. JunB modulates DP5 expression (A-B) INS-1E cells were transfected with pCGJunB plasmid overexpressing JunB3 or control vector (pCG). Western blot was performed to demonstrate JunB overexpression. WT and mutant DP5 reporter activities were measured 24 h after transfection. *P < 0.05 vs. pCG. (C-D) 3T3 junB-/- and 3T3 wild-type mouse fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS. Expression of DP5 was measured by real time RT-PCR and Western blot. *P < 0.05 vs. wild-type.
(A) INS-1E cells were exposed to IL-1β + IFN-γ for 15 min. NF-κB activation was observed by presence of p65 (green) in the cell nucleus using immunofluorescence assay. The primary antibody used was anti-p65 (1:500, Santa Cruz Biotechnology, CA, USA), bar 20m. (B) Nuclear extracts were prepared from INS-1E and DNA binding by nuclear proteins (4 g) and supershift analysis with anti-p65 was performed as previously described.4 The sequence of NF-B consensus oligonucleotide was 5’-AGCTTCAGAGGGGACTTTCCGAGA-3’. The specificity of the protein-DNA complex was assessed by the use of a non radioactive NF-B consensus oligonucleotide, while the specificity of the supershift complex was tested using a non related antibody (-STAT or -IRF-1) (data not shown). The specific NF-B mobility shift bands detected in the X-Ray film were quantified by Biomax 1D image analysis software (Kodak) and expressed as arbitrary units of optical densities (O.D.). The results are expressed as mean + SE. *P < 0.05. (C) The non-degradable NF-κB inhibitor AdIB(SA)2 prevents cytokine-induced NF-κB activation. NF-κB activation was evaluated by immunofluorescence analysis for the presence of its p65 subunit (green) in the nucleus of INS-1E cell infected with a recombinant adenovirus expressing either luciferase (AdLUC) or the IB(SA)2 followed by 30 min exposure to cytokines, bar 20m. (D) Nitrite production measured in medium of AdIκB(SA)2- or AdLuc-infected INS-1E cells treated or not for 24 h with cytokines. *P < 0.05. (E) The iNOS blocker L-NMA significantly reduced cytokine-induced NO formation during a 24 h culture. ***P < 0.001. (F) Percentage of cell death (HO/PI) in INS-1E cells after exposure for 24 h to IL-1β + IFN-γ, the JNK peptide inhibitor (JNKi), L-NMA or combination as indicated. **P < 0.01, ***P < 0.001.
(A) Time course of DP5 and Chop mRNA expression in cytokine-treated INS-1E cells. (B-C) Knockdown of Chop does not prevent cytokine-induced DP5 expression. INS-1E cells were transfected with control or Chop siRNAs and real timer RT-PCR for Chop and DP5 was performed 16 h after cytokine or CPA addition as indicated. *P < 0.05.
1. Lupi R, Dotta F, Marselli L, Del Guerra S, Masini M, Santangelo C et al. Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that β-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated. Diabetes 2002; 51: 1437-1442. 2. Moore F, Colli ML, Cnop M, Esteve MI, Cardozo AK, Cunha DA et al. PTPN2, a candidate gene for type 1 diabetes, modulates interferon- γ-induced pancreatic β-cell apoptosis. Diabetes 2009; 58: 1283-1291. 3. Bakiri L, Lallemand D, Bossy-Wetzel E, Yaniv M. Cell cycle-dependent variations in c-Jun and JunB phosphorylation: a role in the control of cyclin D1 expression. Embo J 2000; 19: 2056-2068. 4. Kutlu B, Darville MI, Cardozo AK, Eizirik DL. Molecular regulation of monocyte chemoattractant protein-1 expression in pancreatic β-cells. Diabetes 2003; 52: 348-355. Yüklə 42,36 Kb. Dostları ilə paylaş:
|