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: Acta Pharmaceutica Sinica. B

Article Title: A new perspective of triptolide-associated hepatotoxicity: the relevance of NF- κ B and NF- κ B-mediated cellular FLICE-inhibitory protein

doi: 10.1016/j.apsb.2020.02.009

Figure Lengend Snippet: Possible role of NF- κ B and NF- κ B-mediated pro-survival signals in TP/LPS-induced hepatotoxicity. (A) Experimental design to detect the time-dependent changes of NF- κ B and its related pro-survival factors. (B)–(G) Relative mRNA levels of NF- κ B target genes, including Ciap1 , Ciap2 , Flip L , Xiap , Tnfaip3 , and Nfkbia , were detected by qPCR with tubulin as the internal control ( n = 6). (H)–(L) Representative Western blots and relative intensity of protein bands of FLIP L , CIAP1, XIAP, and I κ B- α after the treatment of TP and LPS with tubulin as the loading control ( n = 4–6). (M) Representative photomicrographs of liver sections by IHC for P65 1 h after LPS application (200 × ). Scale bar = 50 μm. Results were expressed as mean ± SEM and statistical analysis was performed using Two-way ANOVA following by Tukey's multiple comparison test. *, # , $ P <0.05, **, ## , $$ P <0.01, ***, ### , $$$ P <0.001; ns, no statistical difference.

Article Snippet: Antibody against RIPK1 (17519-1-AP), mixed lineage kinase domain like pseudokinase (MLKL, 66675-1-Ig), myeloperoxidase (MPO, 22225-1-AP), and CIAP1 (10022-1-AP) were purchased from Proteintech (Chicago, IL, USA).

Techniques: Control, Western Blot, Comparison

Journal: Acta Pharmaceutica Sinica. B

Article Title: A new perspective of triptolide-associated hepatotoxicity: the relevance of NF- κ B and NF- κ B-mediated cellular FLICE-inhibitory protein

doi: 10.1016/j.apsb.2020.02.009

Figure Lengend Snippet: The primer sequences used for qPCR assay in mice.

Article Snippet: Antibody against RIPK1 (17519-1-AP), mixed lineage kinase domain like pseudokinase (MLKL, 66675-1-Ig), myeloperoxidase (MPO, 22225-1-AP), and CIAP1 (10022-1-AP) were purchased from Proteintech (Chicago, IL, USA).

Techniques:

Journal: Leukemia

Article Title: Bone marrow stromal cells prevent apoptosis of lymphoma cells by upregulation of anti-apoptotic proteins associated with activation of NF-kappaB (RelB/p52) in non-Hodgkin's lymphoma cells.

doi: 10.1038/sj.leu.2404723

Figure Lengend Snippet: Figure 3 Cell adhesion increases c-IAP1, c-IAP2, and X-IAP protein levels and had no effect on the RNA and protein levels of BCL-2 family members. (a) SUDH-4 cells, in suspension (Sus) versus HS-5 adhesion (HS5-Ad) versus in the conditioned medium (Transwell) analysed by Western blot with c-IAP1, c-IAP2, and x-IAP antibodies. (b) With Bcl-2 and Bcl-xL antibodies. Data shown are representative of three independent experiments.

Article Snippet: The following polyclonal antibodies were purchased: caspases-3, 8, 9, PARP, XIAP, (Cell Signaling Technology, Beverly, MA, USA), c-IAP1, c-IAP2, p50/105, p52/100, RelA, RelB, RelC and NF-kB-inducing kinase (NIK) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), b-actin (Sigma, St Louis, MO, USA).

Techniques: Suspension, Western Blot

Journal: Leukemia

Article Title: Bone marrow stromal cells prevent apoptosis of lymphoma cells by upregulation of anti-apoptotic proteins associated with activation of NF-kappaB (RelB/p52) in non-Hodgkin's lymphoma cells.

doi: 10.1038/sj.leu.2404723

Figure Lengend Snippet: Figure 5 Bortezomib (PS341) sensitizes patient lymphoma cells to mitoxantrone-mediated apoptosis and reduces anti-apopotic protein expression: c-IAP1, c-IAP-2 and XIAP in fresh NHL lymphoma cells adhered to HS-5. (a) Fresh follicular lymphoma cells maintained in suspension (Sus) or adhered to the pre-established monolayer of HS-5 (HS5-Ad) were treated with and without mitoxantrone (2 uM) alone or PS341 (50 nM) alone or mitoxantrone plus PS341 (50 nM) for 2 h and analyzed flow cytometry using Annexin V. (b) Follicular lymphoma (FCL) and (c) DLBL in suspension (Sus) or adhered to the pre-established monolayer of HS-5 (HS5-Ad) or in transwell insert (Transwell) were treated with and without bortezomib (50 nM) for 12 h and the lymphoma cells were collected, and whole cell lysates were analysed analysed by Western blot with c-IAP1, c-IAP2 and XIAP antibodies. Data shown are representative of three independent experiments. DLBL, diffuse Large B cell lymphoma; NHL, non- Hodgkin’s lymphoma.

Article Snippet: The following polyclonal antibodies were purchased: caspases-3, 8, 9, PARP, XIAP, (Cell Signaling Technology, Beverly, MA, USA), c-IAP1, c-IAP2, p50/105, p52/100, RelA, RelB, RelC and NF-kB-inducing kinase (NIK) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), b-actin (Sigma, St Louis, MO, USA).

Techniques: Expressing, Suspension, Cytometry, Western Blot

Journal: Stroke

Article Title: Ischemic Preconditioning Reduces Neurovascular Damage After Hypoxia-Ischemia Via the Cellular Inhibitor of Apoptosis 1 in Neonatal Brain

doi: 10.1161/strokeaha.112.677617

Figure Lengend Snippet: Figure 2. A, The no ischemic preconditioning (IP) group had significantly increased cleaved caspase-8, caspase-9, caspase-3, and poly (ADP-ribose) polymerase (PARP) 24 hours post-hypoxia-ischemia (HI) compared with the IP group. B, The IP group showed significantly increased cellular inhibitor of apoptosis 1 (cIAP1) but not cIAP2, X-linked IAP (XIAP), or survivin. Data from 4 different experiments; *P<0.05.

Article Snippet: Lentivirus-mediated short hairpin RNA targeting cIAP1 mRNA (LV-sh-cIAP1; sc-29848-V; Santa Cruz) and nontarget shRNA control lentiviral particles encoding a scrambled shRNA sequence (LV-sh-scramble control; sc-108080) were used.

Techniques:

Journal: Stroke

Article Title: Ischemic Preconditioning Reduces Neurovascular Damage After Hypoxia-Ischemia Via the Cellular Inhibitor of Apoptosis 1 in Neonatal Brain

doi: 10.1161/strokeaha.112.677617

Figure Lengend Snippet: Figure 3. A, Immunohistochemistry showed that cellular inhibitor of apoptosis 1 (cIAP1) was markedly decreased in the group with no ischemic preconditioning (IP) but increased in the IP group 24-hour post-hypoxia-ischemia (HI). In the IP group, cIAP1 was mainly expressed in the vascular (arrows) and nonvascular cells. Scale bar, 100 μm. B, Immunofluorescence confirmed that cIAP1 expressed mainly in the rat endothelial cell antigen-1-positive endothelial cells and NeuN-positive neurons, but not in astrocytes (glial fibrillary acidic protein [GFAP]), in IP group. C, Compared with control, cIAP1 was significantly upregulated in IP group pretreated with control small interfering RNA (siRNA) but not in that pretreated with cIAP1 siRNA. n=4 per group. D, In IP group, the pups pretreated with cIAP1 siRNA had significantly more brain damage than those with control siRNA. n=7 per group. Scale bar, 20 μm. *P<0.05; **P<0.01; #P<0.001.

Article Snippet: Lentivirus-mediated short hairpin RNA targeting cIAP1 mRNA (LV-sh-cIAP1; sc-29848-V; Santa Cruz) and nontarget shRNA control lentiviral particles encoding a scrambled shRNA sequence (LV-sh-scramble control; sc-108080) were used.

Techniques: Immunohistochemistry, Immunofluorescence, Control, Small Interfering RNA

Journal: Stroke

Article Title: Ischemic Preconditioning Reduces Neurovascular Damage After Hypoxia-Ischemia Via the Cellular Inhibitor of Apoptosis 1 in Neonatal Brain

doi: 10.1161/strokeaha.112.677617

Figure Lengend Snippet: Figure 4. SH-SY5Y neurons. A, Left, Compared with controls, cytotoxicity increased progressively when oxygen- glucose deprivation (OGD) duration increased to 15 hours (left). Right, Eight- hour OGD preconditioning before 15-hour OGD significantly decreased cytotoxicity. B, Preconditioned cells had significantly increased cellular inhibitor of apoptosis 1 (cIAP1) at 1, 6, and 24 hours post-OGD than nonpreconditioned cells. C, Lentivi rus-mediated short hairpin RNA targeting cIAP1 mRNA (LV-sh-cIAP1) cells had 25% of the cIAP1 levels of LV-sh-scram ble cells. D, Post-OGD, preconditioned LV-sh-scramble cells were significantly less cytotoxic than nonpreconditioned LV-sh-scramble cells, and preconditioned LV-sh-cIAP1 cells had significantly more cytotoxicity than preconditioned LV- sh-scramble cells. Data were from 4 different experiments. *P<0.05; #P<0.001.

Article Snippet: Lentivirus-mediated short hairpin RNA targeting cIAP1 mRNA (LV-sh-cIAP1; sc-29848-V; Santa Cruz) and nontarget shRNA control lentiviral particles encoding a scrambled shRNA sequence (LV-sh-scramble control; sc-108080) were used.

Techniques: shRNA

Journal: Stroke

Article Title: Ischemic Preconditioning Reduces Neurovascular Damage After Hypoxia-Ischemia Via the Cellular Inhibitor of Apoptosis 1 in Neonatal Brain

doi: 10.1161/strokeaha.112.677617

Figure Lengend Snippet: Figure 5. A, Left, Cytotoxicity increased progressively in human microvascular endothelial cell-1 (HMEC-1) endothelial cells when oxygen–glucose deprivation (OGD) duration increased up to 15 hours. Right, Seven-hour OGD precondition ing before 15-hour OGD significantly decreased cytotoxicity in HMEC-1 cells. B, Preconditioned cells had significantly more cellular inhibitor of apoptosis 1 (cIAP1) than nonpreconditioned cells at 24 and 48 hours post-OGD. C, Lentivirus- mediated short hairpin RNA targeting cIAP1 mRNA (LV-sh-cIAP1) cells had 45% of cIAP1 levels of LV-sh-scramble cells. D, Post-OGD, preconditioned LV- sh-cIAP1 cells had significantly more cytotoxicity than preconditioned LV- sh-scramble cells. Data from 4 different experiments. *P<0.05; #P<0.001.

Article Snippet: Lentivirus-mediated short hairpin RNA targeting cIAP1 mRNA (LV-sh-cIAP1; sc-29848-V; Santa Cruz) and nontarget shRNA control lentiviral particles encoding a scrambled shRNA sequence (LV-sh-scramble control; sc-108080) were used.

Techniques: shRNA

Journal: Stroke

Article Title: Ischemic Preconditioning Reduces Neurovascular Damage After Hypoxia-Ischemia Via the Cellular Inhibitor of Apoptosis 1 in Neonatal Brain

doi: 10.1161/strokeaha.112.677617

Figure Lengend Snippet: Figure 6. A, Lentiviruses encoding cel lular inhibitor of apoptosis 1 (LV-cIAP1) SH-SY5Y neurons had 30% increases in cIAP1 compared with LV-control neurons. B, After oxygen–glucose depri vation (OGD), LV-cIAP1 neurons had significantly decreased cytotoxicity than LV-control neurons. C, LV-cIAP1 human microvascular endothelial cells-1 (HMEC-1) showed 70% increases in cIAP1 compared with LV-control cells. D, Post-OGD, LV-cIAP1 cells had sig nificantly decreased cytotoxicity than LV-control cells. Data from 4 different experiments. **P<0.01; #P<0.001.

Article Snippet: Lentivirus-mediated short hairpin RNA targeting cIAP1 mRNA (LV-sh-cIAP1; sc-29848-V; Santa Cruz) and nontarget shRNA control lentiviral particles encoding a scrambled shRNA sequence (LV-sh-scramble control; sc-108080) were used.

Techniques: Control

Journal: Journal of Biological Chemistry

Article Title: Cellular Inhibitor of Apoptosis Protein 1 (cIAP1) Stability Contributes to YM155 Resistance in Human Gastric Cancer Cells

doi: 10.1074/jbc.m114.600874

Figure Lengend Snippet: FIGURE 3. Double knockdown of survivin and cIAP1 induces cell death in YM155-resistant MKN45 cells. A and B, KATOIII (A) and MKN45 (B) cells were co-transfected with survivin-siRNA and cIAP1 siRNA for 48 h, and then cell death was determined using the trypan blue exclusion method. Cell lysates were subjected to Western blot analysis with anti-survivin and anti-CIAP1 and the loading control -actin. The data are the mean S.D.; **, p 0.01.

Article Snippet: Plasmids, siRNAs, and Transfections—Survivin and cIAP1 cDNAs were purchased from Origene (Rockville, MD).

Techniques: Knockdown, Transfection, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Cellular Inhibitor of Apoptosis Protein 1 (cIAP1) Stability Contributes to YM155 Resistance in Human Gastric Cancer Cells

doi: 10.1074/jbc.m114.600874

Figure Lengend Snippet: FIGURE 6. The survivin heteromer decreases after YM155 in YM155-sensitive KATOIII cells but not MKN45 cells. A, MKN45 cells were immunoprecipi- tated with anti-survivin or anti-cIAP1 or anti-rabbit IgG antibodies. Interaction of survivin and cIAP1 was determined with Western blot analysis using anti-survivin or anti-cIAP1 antibodies. B, KATOIII and MKN45 cells were treated with YM155 (20 nM) for 48 h. Cell lysates were run on a native gel. Various heteromers of survivin were determined with Western blot analysis using anti-survivin antibody. C, KATOIII and MKN45 cells were treated with YM155 (20 nM) for 48 h and then cell lysates were immunoprecipitated (IP) using an anti-survivin antibody. Determination of the binding of survivin to the monomer or dimer of cIAP1 was determined via native PAGE using anti-cIAP1.

Article Snippet: Plasmids, siRNAs, and Transfections—Survivin and cIAP1 cDNAs were purchased from Origene (Rockville, MD).

Techniques: Western Blot, Immunoprecipitation, Binding Assay, Clear Native PAGE