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

Journal: Journal of Biological Chemistry

Article Title: Critical Role for Antiapoptotic Bcl-xL and Mcl-1 in Human Macrophage Survival and Cellular IAP1/2 (cIAP1/2) in Resistance to HIV-Vpr-induced Apoptosis

doi: 10.1074/jbc.m111.312660

Figure Lengend Snippet: FIGURE 6. Vpr down-regulates the expression of Bcl2 and cIAP1 in monocytic cells. Primary monocytes (2 106/ml) (A) and THP1 cells (1 106/ml) were treated with the indicated concentrations of Vpr or mVpr for 24 h (B) and 12 h (C). Total cell proteins were subjected to Western blotting, and the membranes were probed with the indicated antibodies. In THP1 cells, the expression level of antiapoptotic proteins was quantified to control for equal protein loading. Based on the densitometric analysis, the bar graphs in the bottom panels show the mean S.D. (error bars) of at least three experiments. *, p 0.05.

Article Snippet: Transfection with siRNA—THP1-MACs (5 105/ml) were treated with cIAP1 siRNA, cIAP2 siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and control siRNA (Qiagen, Venlo, The Netherlands) in serum-free medium, using Fugene 6 (Roche Applied Science) as a transfection reagent, according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Critical Role for Antiapoptotic Bcl-xL and Mcl-1 in Human Macrophage Survival and Cellular IAP1/2 (cIAP1/2) in Resistance to HIV-Vpr-induced Apoptosis

doi: 10.1074/jbc.m111.312660

Figure Lengend Snippet: FIGURE 7. Vpr does not affect the expression of Bcl2 and cIAP1 in macrophages. MDMs (A) and THP1-MACs (B) were treated with the indicated concen- trations of Vpr or mVpr for 24 h. Total cell proteins were subjected to Western blotting, and the membranes were probed with specific antibodies against various members of the Bcl2 and IAP families. The results shown are representative of three separate experiments with similar results.

Article Snippet: Transfection with siRNA—THP1-MACs (5 105/ml) were treated with cIAP1 siRNA, cIAP2 siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and control siRNA (Qiagen, Venlo, The Netherlands) in serum-free medium, using Fugene 6 (Roche Applied Science) as a transfection reagent, according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Critical Role for Antiapoptotic Bcl-xL and Mcl-1 in Human Macrophage Survival and Cellular IAP1/2 (cIAP1/2) in Resistance to HIV-Vpr-induced Apoptosis

doi: 10.1074/jbc.m111.312660

Figure Lengend Snippet: FIGURE 10. IAPs protect macrophages against Vpr-induced apoptosis. A, after differentiation, THP1-MACs (5 105/ml) were transfected with cIAP1, cIAP2, or control siRNA as described under “Experimental Procedures.” Following transfection, cells were collected after 24 h for evaluation of protein knockdown. Total cell proteins were subjected to Western blotting, and the membranes were probed with antibodies specific for cIAP1, cIAP2, and Bcl2 to ensure siRNA specificity. B and C, on the second day after transfection, cells were treated with Vpr (2.5 M) for another 24 h and stained with PI for apoptosis measurement (B) or subjected to Western blotting for PARP and caspase-3 cleavage (C). Only 500 ng of IAP siRNA and control siRNA were used for these experiments. The bar graph in the top panel of B shows the mean percentage of apoptosis S.D. (error bars) of four separate experiments. *, p 0.05. Histograms in the bottom panel of B show one representative experiment indicating the percentage of cells with subdiploid DNA content.

Article Snippet: Transfection with siRNA—THP1-MACs (5 105/ml) were treated with cIAP1 siRNA, cIAP2 siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and control siRNA (Qiagen, Venlo, The Netherlands) in serum-free medium, using Fugene 6 (Roche Applied Science) as a transfection reagent, according to the manufacturer’s instructions.

Techniques: Transfection, Control, Knockdown, Western Blot, Staining

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