Journal: PLOS ONE

Article Title: Differential regulation of BIRC2 and BIRC3 expression by inflammatory cytokines and glucocorticoids in pulmonary epithelial cells

doi: 10.1371/journal.pone.0286783

Figure Lengend Snippet: (A-B) A549 cells, (C) pHBEC-SC or (D) pHBEC-ALI were not stimulated (NS) or treated with IL1B (1 ng/ml), TNF (10 ng/ml), and/or dexamethasone ( Dex ; 1 μM) or budesonide ( Bud ; 300 nM). Cells from N = 4–5 experiments were harvested at the indicated times for qPCR analysis. (B) The sum of the effects ( i . e ., fold– 1) for TNF and Dex ( sum ( ΔTNF + ΔDex) ) and the effect (fold– 1) of the combination treatment ( comboΔ(TNF + Dex) ) at 6 and 24 h is plotted. (C & D) The sum of the effects of IL1B and Bud ( sum(ΔIL1B + ΔBud) ) and the effect (fold– 1) of the combination treatment ( comboΔ(IL1B+Bud) ) at 6 h is plotted. Data for BIRC3 and BIRC2 were normalized to GAPDH, expressed as fold of NS either at t = 1 h (A), or at 6 h (C & D) and are plotted as mean ±SE. Significance was tested using one-way ANOVA with a Dunnett’s post-hoc test in A , and a Tukey’s post-hoc test in C & E . Significance in B , C and D was tested by paired t test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 indicates significance relative to NS or as otherwise indicated.

Article Snippet: Proteins were then transferred to nitrocellulose membranes before blocking and incubation with primary antibodies against BIRC3 (cIAP2 #3130; Cell Signaling Technologies, Danvers, MA), BIRC2 (cIAP1 #7065; Cell Signaling Technologies), RELA (p65 sc-8008; SantaCruz, Dallas, TX), GR (PA1-511A; Thermo Fisher Scientific), K48-linkage specific polyubiquitin (#8081; Cell Signaling Technologies), GAPDH (MCA4739; Bio-Rad, Hercules, CA) overnight at 4°C.

Techniques:

Journal: PLOS ONE

Article Title: Differential regulation of BIRC2 and BIRC3 expression by inflammatory cytokines and glucocorticoids in pulmonary epithelial cells

doi: 10.1371/journal.pone.0286783

Figure Lengend Snippet: (A-B) A549 cells, (C-D) pHBEC-SC or pHBEC-ALI were not stimulated (NS) or treated with IL1B (1 ng/ml), TNF (10 ng/ml), and/or dexamethasone ( Dex ; 1 μM) or budesonide ( Bud ; 300 nM). Cells were harvested at 6 and 24 h for western blot analysis. (B) The sum of the effects ( i . e ., fold– 1) for TNF and Dex ( sum ( ΔTNF + ΔDex) ) and the effect (fold– 1) of the combination treatment ( comboΔ(TNF + Dex) ) from A , is plotted. (D) The sum of the effects ( i . e ., fold– 1) of IL1B and Bud ( sum(ΔIL1B + ΔBud) ) and the effect (fold– 1) of the combination treatment ( comboΔ(IL1B+Bud) ) from C , is plotted. Blots representative of N = 4–11 experiments are shown. Data for BIRC3 and BIRC2 were normalized to GAPDH and plotted as mean ±SE. Significance was tested using one-way ANOVA with a Tukey’s post-hoc test in A and C . Significance in B and D was tested by paired t test * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 indicates significance relative to NS or as otherwise indicated.

Article Snippet: Proteins were then transferred to nitrocellulose membranes before blocking and incubation with primary antibodies against BIRC3 (cIAP2 #3130; Cell Signaling Technologies, Danvers, MA), BIRC2 (cIAP1 #7065; Cell Signaling Technologies), RELA (p65 sc-8008; SantaCruz, Dallas, TX), GR (PA1-511A; Thermo Fisher Scientific), K48-linkage specific polyubiquitin (#8081; Cell Signaling Technologies), GAPDH (MCA4739; Bio-Rad, Hercules, CA) overnight at 4°C.

Techniques: Western Blot

Journal: PLOS ONE

Article Title: Differential regulation of BIRC2 and BIRC3 expression by inflammatory cytokines and glucocorticoids in pulmonary epithelial cells

doi: 10.1371/journal.pone.0286783

Figure Lengend Snippet: (A) A549 cells harbouring the NF-κB-dependent reporter, 6κBtkluc.neo, were not stimulated (NS) or treated with IL1B (1 ng/ml), with or without prior incubation with increasing concentrations of control siRNAs (siCTL) or RELA-targeting siRNAs (siRELA) for 48 h (upper panel). (B) NF-κB-dependent reporter cells were not stimulated (NS) or treated with IL1B (1 ng/ml), or TNF (10 ng/ml), with or without prior incubation with siCTL or siRELA (both at 1 nM) for 48 h. After 6 h, cells were harvested for luciferase activity determination and data are plotted as fold of NS ±SE. (C) As in A , A549 cells were incubated with or without siCTL or siRELA, each at 1 nM, prior to stimulation with IL1B (1 ng/ml) or IL1B-plus-dexamethasone at 1 μM (IL+D). Cells were harvested at 6 h for western blot analysis of BIRC3, BIRC2 (data not shown), RELA and GAPDH. Representative blots are shown. (D) A549 cells were either not infected, or infected with Ad5-IκBαΔN or Ad5-GFP at an MOI of 25, before treatment with IL1B or IL+D. Cells were harvested at 6 h for qPCR analysis of BIRC2, BIRC3 and GAPDH. In C and D , mRNA or protein data for BIRC3, BIRC2 or RELA were normalized to GAPDH and are plotted as mean ± SE. Data from N = 4–5 experiments are shown, and significance was tested in panels A , C and D using one-way ANOVA with a Tukey’s post-hoc test. Significance in B was tested by paired t test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 indicates significance relative to NS or as otherwise indicated.

Article Snippet: Proteins were then transferred to nitrocellulose membranes before blocking and incubation with primary antibodies against BIRC3 (cIAP2 #3130; Cell Signaling Technologies, Danvers, MA), BIRC2 (cIAP1 #7065; Cell Signaling Technologies), RELA (p65 sc-8008; SantaCruz, Dallas, TX), GR (PA1-511A; Thermo Fisher Scientific), K48-linkage specific polyubiquitin (#8081; Cell Signaling Technologies), GAPDH (MCA4739; Bio-Rad, Hercules, CA) overnight at 4°C.

Techniques: Incubation, Control, Luciferase, Activity Assay, Western Blot, Infection

Journal: PLOS ONE

Article Title: Differential regulation of BIRC2 and BIRC3 expression by inflammatory cytokines and glucocorticoids in pulmonary epithelial cells

doi: 10.1371/journal.pone.0286783

Figure Lengend Snippet: (A) A549 cells were pretreated, or not, with PS-1145 (30 μM) for 90 min prior to addition of IL1B (1 ng/ml), TNF (10 ng/ml), dexamethasone ( Dex ; 1 μM) or in combination (IL+D, T+D), as indicated. Cells were harvested at 6 h for qPCR analysis. (B) A549 cells were pretreated, or not, with PS-1145 (30 μM) or ML-120B (30 μM) for 90 min prior to treatment with IL1B (1 ng/ml) or TNF (10 ng/ml). Cells were harvested at 6 and 24 h for western blot analysis. Representative blots are shown. A & B , BIRC2 and BIRC3 mRNA or protein (BIRC2 data not shown) data, from N = 5–10 experiments, were normalized to GAPDH and plotted as mean ±SE. Significance was tested using one-way ANOVA with a Tukey’s post-hoc test in A , and Bonferroni’s post-hoc test in B . * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001 indicates significance relative to NS or as otherwise indicated.

Article Snippet: Proteins were then transferred to nitrocellulose membranes before blocking and incubation with primary antibodies against BIRC3 (cIAP2 #3130; Cell Signaling Technologies, Danvers, MA), BIRC2 (cIAP1 #7065; Cell Signaling Technologies), RELA (p65 sc-8008; SantaCruz, Dallas, TX), GR (PA1-511A; Thermo Fisher Scientific), K48-linkage specific polyubiquitin (#8081; Cell Signaling Technologies), GAPDH (MCA4739; Bio-Rad, Hercules, CA) overnight at 4°C.

Techniques: Western Blot

Journal: PLOS ONE

Article Title: Differential regulation of BIRC2 and BIRC3 expression by inflammatory cytokines and glucocorticoids in pulmonary epithelial cells

doi: 10.1371/journal.pone.0286783

Figure Lengend Snippet: ( A-B ) A549 cells were either not treated or pre-treated with ORG35417 (1 μM) for 1 h before addition of dexamethasone (Dex) at the indicated concentrations. Cells were harvested at 6 h for; A , qPCR and Schild analysis, or B , western blot analysis. ( C-D ) A549 cells were incubated with either control siRNAs (siCTL) or GR-targeting siRNAs (siGR) for 48 h prior to addition of dexamethasone ( Dex ; 1 μM), IL1B (1 ng/ml), or IL1B plus dexamethasone (IL+D). Cells were harvested at 6 h for western blot analysis of BIRC3, GR and GAPDH. Representative blots are shown. BIRC3 and GR mRNA or protein data, from N = 5–8 experiments, were normalized to GAPDH and plotted as mean ±SE. Significance was tested using one-way ANOVA with a Tukey’s post-hoc test. * p ≤ 0.05 and ** p ≤ 0.01 indicates significance relative to NS or as otherwise indicated.

Article Snippet: Proteins were then transferred to nitrocellulose membranes before blocking and incubation with primary antibodies against BIRC3 (cIAP2 #3130; Cell Signaling Technologies, Danvers, MA), BIRC2 (cIAP1 #7065; Cell Signaling Technologies), RELA (p65 sc-8008; SantaCruz, Dallas, TX), GR (PA1-511A; Thermo Fisher Scientific), K48-linkage specific polyubiquitin (#8081; Cell Signaling Technologies), GAPDH (MCA4739; Bio-Rad, Hercules, CA) overnight at 4°C.

Techniques: Western Blot, Incubation, Control

Journal: PLOS ONE

Article Title: Differential regulation of BIRC2 and BIRC3 expression by inflammatory cytokines and glucocorticoids in pulmonary epithelial cells

doi: 10.1371/journal.pone.0286783

Figure Lengend Snippet: A549 cells were not stimulated (NS) or treated with IL1B (1 ng/ml), or TNF (10 ng/ml) for 1 h prior to the addition (t = 0) of cycloheximide (CHX; 10 μg/ml), as indicated. Cells were harvested: ( A ) at t = 0; and, ( B ) at t = 0, 0.5, 1, 2 and 6 h for western blot analysis of BIRC3, BIRC2 and GAPDH. Representative blots are shown for each panel. In A , densitometric data for BIRC2 and BIRC3 were normalized to GAPDH. In B , densitometric data, normalized to GAPDH, were expressed as a percentage of each treatment at t = 0. Data from N = 5 experiments are plotted as mean ±SE. Significance in A , was tested by paired t test and, in B , by one-way ANOVA with a Dunnett’s post-hoc test. * p ≤ 0.05 in A indicates significance relative to NS or in B , relative to NS (t = 0).

Article Snippet: Proteins were then transferred to nitrocellulose membranes before blocking and incubation with primary antibodies against BIRC3 (cIAP2 #3130; Cell Signaling Technologies, Danvers, MA), BIRC2 (cIAP1 #7065; Cell Signaling Technologies), RELA (p65 sc-8008; SantaCruz, Dallas, TX), GR (PA1-511A; Thermo Fisher Scientific), K48-linkage specific polyubiquitin (#8081; Cell Signaling Technologies), GAPDH (MCA4739; Bio-Rad, Hercules, CA) overnight at 4°C.

Techniques: Western Blot

Journal: PLOS ONE

Article Title: Differential regulation of BIRC2 and BIRC3 expression by inflammatory cytokines and glucocorticoids in pulmonary epithelial cells

doi: 10.1371/journal.pone.0286783

Figure Lengend Snippet: A549 cells were not stimulated (NS) or treated with IL1B (1 ng/ml), or TNF (10 ng/ml) for 6 h prior to the addition (t = 0) of cycloheximide (CHX; 10 μg/ml), as indicated. Cells were harvested: ( A ) at t = 0; and, ( B ) at t = 0, 0.5, 1, 2 and 6 h for western blot analysis of BIRC3 and GAPDH. Representative blots are shown for each panel. In A , densitometric data for BIRC3 were normalized to GAPDH. In B , densitometric data, normalized to GAPDH, were expressed as a percentage of each treatment at t = 0. Data from N = 5 experiments are plotted as mean ±SE. Significance in A , was tested by paired t test and, in B , by one-way ANOVA with a Dunnett’s post-hoc test. ** p ≤ 0.01 and *** p ≤ 0.001 in A indicates significance relative to NS.

Article Snippet: Proteins were then transferred to nitrocellulose membranes before blocking and incubation with primary antibodies against BIRC3 (cIAP2 #3130; Cell Signaling Technologies, Danvers, MA), BIRC2 (cIAP1 #7065; Cell Signaling Technologies), RELA (p65 sc-8008; SantaCruz, Dallas, TX), GR (PA1-511A; Thermo Fisher Scientific), K48-linkage specific polyubiquitin (#8081; Cell Signaling Technologies), GAPDH (MCA4739; Bio-Rad, Hercules, CA) overnight at 4°C.

Techniques: Western Blot

Journal: PLOS ONE

Article Title: Differential regulation of BIRC2 and BIRC3 expression by inflammatory cytokines and glucocorticoids in pulmonary epithelial cells

doi: 10.1371/journal.pone.0286783

Figure Lengend Snippet: (A) As depicted in the schematic, A549 cells were either not stimulated (NS) or treated with IL1B (1 ng/ml), or TNF (10 ng/ml) for 1 h before the addition (t = 0) of MG-132 (10 μM), MG-262 (10 μM), PR-171 (10 μM) or E-64 (10 μM) either in the absence or presence of cycloheximide (CHX; 10 μg/ml). All cells were harvested at t = 6 h for western blot analysis. (B) Shows the effects of NS, IL1B and TNF and the inhibitors in the absence of CHX. (C) Shows NS or TNF in absence or presence of CHX along with the further effect of the inhibitors. Representative blots are shown and densitometric data for BIRC3 and BIRC2, normalized to GAPDH, and are plotted as mean ±SE. Data from N = 5–6 experiments are shown and significance tested using one-way ANOVA with a Tukey’s post-hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 indicates significance relative to NS or as otherwise indicated.

Article Snippet: Proteins were then transferred to nitrocellulose membranes before blocking and incubation with primary antibodies against BIRC3 (cIAP2 #3130; Cell Signaling Technologies, Danvers, MA), BIRC2 (cIAP1 #7065; Cell Signaling Technologies), RELA (p65 sc-8008; SantaCruz, Dallas, TX), GR (PA1-511A; Thermo Fisher Scientific), K48-linkage specific polyubiquitin (#8081; Cell Signaling Technologies), GAPDH (MCA4739; Bio-Rad, Hercules, CA) overnight at 4°C.

Techniques: Western Blot

Journal: bioRxiv

Article Title: ZFAND6 is a subunit of a TRAF2-cIAP E3 ubiquitin ligase complex essential for mitophagy

doi: 10.1101/2024.03.27.586763

Figure Lengend Snippet: A) Confocal microscopy with fluorescently conjugated antibodies to pUb using WT and Zfand6 −/− BMDMs treated with 5 μM oligomycin A and 1 μM antimycin A for 3 hr. in the presence of 100 μM leupeptin. Scale bars represent 5 μm. Data presented as mean ± SEM. ( B, C) Co-IP assays were performed with lysates from 293T cells transfected with the indicated plasmids. Immunoprecipitations and western blotting were performed with the indicated antibodies to examine ZFAND6-TRAF2 (B) and ZFAND6-cIAP1 (C) interactions. (D) Co-IP assay was performed with lysates from WT and Zfand6 −/− BMDMs treated with 5 μM oligomycin A and 1 μM antimycin A for the indicated times. IPs were performed with either isotype control antibody or TRAF2 antibody. Western blotting was performed with the indicated antibodies. Unpaired Student’s t-test. ****P<0.0001.

Article Snippet: Antibodies used for western blotting are: STING Rabbit mAb (Cell Signaling Cat# 13647S), Phospho-STING rabbit anti-mouse mAb (Cell Signaling Cat# 72971S), IRF3 rabbit mAb (Cell Signaling Cat# 4302S), p-IRF3 rabbit mAb (Cell Signaling Cat# 4947S), TBK1 rabbit mAb (Cell Signaling Cat# 3504S), p-TBK1 rabbit mAb (Cell Signaling Cat# 5483S), TRAF2 rabbit pAb (Proteintech Cat# 26846-1-AP), cIAP1 Rabbit pAb (Proteintech Cat# 10022-1-AP), HA rabbit pAb (Proteintech Cat# 51064-2-AP), MTCO2 Rabbit pAb (Proteintech Cat# 55070-1-AP), GAPDH mouse mAb (Proteintech Cat# 60004-1-IG), Flag mouse mAb (Proteintech Cat# 66008-4-Ig), Myc Mouse mAb (Proteintech Cat# 60003-2-Ig) and ZFAND6 rabbit pAb (Origene Cat# TA335508).

Techniques: Confocal Microscopy, Co-Immunoprecipitation Assay, Transfection, Western Blot, Control

Journal: International journal of cardiology

Article Title: Enhanced external counterpulsation inhibits endothelial apoptosis via modulation of BIRC2 and Apaf-1 genes in porcine hypercholesterolemia.

doi: 10.1016/j.ijcard.2013.11.033

Figure Lengend Snippet: Fig. 6. Positive immunostaining of Apaf-1 and BIRC2 in isolated aortic endothelial cells (as indicated by arrow). Representative immunocytochemical photomicrographs (×400) of Apaf-1 and BIRC2 in isolated aortic endothelial cells were shown above and quantitative analysis of Apaf-1 and BIRC2 expression was made. The protein expression of Apaf-1 gene was increased remarkably in the HC group but significantly declined after EECP treatment (bar graph, top). However, the BIRC2 expression was significantly restored by EECP (bar graph, bottom). Data were expressed as mean ± SD; #P b 0.05 compared to Normal; *P b 0.05 compared with group HC.

Article Snippet: Sections were incubated with rabbit monoclonal Cleaved Caspase-3 antibody (at 1:400 dilution; Cell Signaling Technology, Inc. Danvers, MA, USA), rabbit polyclonal Apaf1 antibody (at 1:200 dilution; Labvision Corp, Fremont, CA, USA) and goat polyclonal BIRC2 antibody (at 1:200 dilution; R&D Systems Inc, Minneapolis, MN, USA).

Techniques: Immunostaining, Isolation, Expressing

Journal: International journal of cardiology

Article Title: Enhanced external counterpulsation inhibits endothelial apoptosis via modulation of BIRC2 and Apaf-1 genes in porcine hypercholesterolemia.

doi: 10.1016/j.ijcard.2013.11.033

Figure Lengend Snippet: Fig. 7. Transcriptional expression of Apaf-1 and BIRC2 genes in isolated aortic endothelial cells. Representative electrophoresis bands of RT-PCR products of both genes Apaf-1 and BIRC2 were presented (top) and quantitatively analyzed by comparing with the internal standard β-actin (bottom). Relative mRNA expression of Apaf-1gene was increased significantly in the HC group but declined after EECP. However, mRNA expression of BIRC2 gene was decreased remarkably in the HC group; EECP could significantly restore its expression. Data were expressed as mean ± SD; #P b 0.05 compared to Normal; *P b 0.05 compared with group HC.

Article Snippet: Sections were incubated with rabbit monoclonal Cleaved Caspase-3 antibody (at 1:400 dilution; Cell Signaling Technology, Inc. Danvers, MA, USA), rabbit polyclonal Apaf1 antibody (at 1:200 dilution; Labvision Corp, Fremont, CA, USA) and goat polyclonal BIRC2 antibody (at 1:200 dilution; R&D Systems Inc, Minneapolis, MN, USA).

Techniques: Expressing, Isolation, Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TRAIL and Taurolidine induce apoptosis and decrease proliferation in human fibrosarcoma

doi: 10.1186/1756-9966-27-82

Figure Lengend Snippet: Additional information about the genes whose expression was changed more than two-fold in the experiments.

Article Snippet: To validate the findings of changed gene expression on protein level, Western Blots were performed using an SDS-page gel and the following antibodies (rabbit): Rho GDIα/ARGHGDIA (C-21), GADD 45α (H-165), c-IAP2 / BIRC3 (H-85), GADD 34/PPP1R15A (S-20), NIK/MAP3K14 (H-248), and IκB-α/NFKBIA (C-21), purchased from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TRAIL and Taurolidine induce apoptosis and decrease proliferation in human fibrosarcoma

doi: 10.1186/1756-9966-27-82

Figure Lengend Snippet:

Article Snippet: To validate the findings of changed gene expression on protein level, Western Blots were performed using an SDS-page gel and the following antibodies (rabbit): Rho GDIα/ARGHGDIA (C-21), GADD 45α (H-165), c-IAP2 / BIRC3 (H-85), GADD 34/PPP1R15A (S-20), NIK/MAP3K14 (H-248), and IκB-α/NFKBIA (C-21), purchased from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Reverse Transcription Polymerase Chain Reaction, Microarray, TaqMan Assay, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: TRAIL and Taurolidine induce apoptosis and decrease proliferation in human fibrosarcoma

doi: 10.1186/1756-9966-27-82

Figure Lengend Snippet: Summary of the expression changes of apoptosis related genes for the single substances (TRD 250 μmol/l, TRAIL 50 ng/ml) compared to untreated cells and the combination therapy compared to Control, TRD and TRAIL treated cells.

Article Snippet: To validate the findings of changed gene expression on protein level, Western Blots were performed using an SDS-page gel and the following antibodies (rabbit): Rho GDIα/ARGHGDIA (C-21), GADD 45α (H-165), c-IAP2 / BIRC3 (H-85), GADD 34/PPP1R15A (S-20), NIK/MAP3K14 (H-248), and IκB-α/NFKBIA (C-21), purchased from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Expressing, Control, Inhibition, Virus, Activation Assay, Sequencing, Cell Differentiation, Activity Assay