Journal: Human Gene Therapy

Article Title: Small Interfering RNA Targeting Heme Oxygenase-1 (HO-1) Reinforces Liver Apoptosis Induced by Ischemia-Reperfusion Injury in Mice: HO-1 Is Necessary for Cytoprotection

doi: 10.1089/hum.2009.049

Figure Lengend Snippet: (A) Western blot analysis of HO-1 protein expression after HO-1 siRNA addition to CoPP-treated macrophages. RAW 264.7 macrophages were transfected with two different HO-1 siRNA sequences (1 and 2) and nonspecific control siRNA before CoPP treatment. Lanes 1 and 2 represent HO-1 siRNA sequences 1 and 2, respectively. Note: HO-1 siRNA sequence 2 inhibited CoPP-induced HO-1 protein induction, whereas sequence 1 and nonspecific siRNA had no effect on HO-1 expression. (B) Western blot analysis of HO-1 and caspase-3 gene products in Ad-HO-1-transfected YPEN-1 cells. The expression of HO-1 and caspase-3 was probed with rabbit anti-mouse HO-1 (a) and caspase-3 (b) antibodies. Lane 1, YPEN-1 cells alone; lane 2, YPEN-1 cells plus etoposide (50 μM); lane 3, YPEN-1 cells transfected with Ad-HO-1 and HO-1 siRNA plus etoposide (50 μM); lane 4, YPEN-1 cells transfected with Ad-HO-1 and nonspecific siRNA plus etoposide (50 μM); lane 5, YPEN-1 cells transfected with Ad-HO-1; lane 6, YPEN-1 cells transfected with Ad-β-gal. Note the selectively inhibited expression of HO-1 in HO-1 siRNA-treated YPEN-1 cells (lane 3a), as compared with nonspecific siRNA and Ad-HO-1 (lanes 4a and 5a). In contrast, the expression of caspase-3 increased in cells treated with 50 μM etoposide (lane 2b) or after HO-1 siRNA (lane 3b) or Ad-β-gal (lane 6b), as compared with nonspecific siRNA (lane 4b) or Ad-HO-1 (lane 5b). Anti-β-actin antibody was used to ensure equal protein amounts between the samples. Data shown are representative of three separate experiments.

Article Snippet: The murine nonspecific siRNA scrambled duplex (sense, 5′-GCGCGCUUUGUAGGAUUCG-3′; antisense, 5′-CGAAUCCUACAAAGCGCGC-3′) and the nonsilencing siRNA (NS siRNA: sense, 5′-UUCUCCGAACGUGUCACGU-3′; antisense, 5′-ACGUGACACGUUCGGAGAA-3′) were also synthesized (Qiagen, Chatsworth, CA), and served as negative controls.

Techniques: Western Blot, Expressing, Transfection, Control, Sequencing

Journal: Human Gene Therapy

Article Title: Small Interfering RNA Targeting Heme Oxygenase-1 (HO-1) Reinforces Liver Apoptosis Induced by Ischemia-Reperfusion Injury in Mice: HO-1 Is Necessary for Cytoprotection

doi: 10.1089/hum.2009.049

Figure Lengend Snippet: Hepatocellular damage, as analyzed by sGOT level (IU/liter), in mice that underwent 90 min of hepatic warm ischemia followed by 6 hr of reperfusion. Note: sGOT levels were significantly increased in recipients treated with HO-1 siRNA, as compared with control scrambled siRNA, nonspecific siRNA, or Ad-HO-1 (*p < 0.05). In contrast, Ad-HO-1 decreased sGOT levels, as compared with Ad-β-gal controls (*p < 0.05). Mean and SD are shown (n = 4–6 per group).

Article Snippet: The murine nonspecific siRNA scrambled duplex (sense, 5′-GCGCGCUUUGUAGGAUUCG-3′; antisense, 5′-CGAAUCCUACAAAGCGCGC-3′) and the nonsilencing siRNA (NS siRNA: sense, 5′-UUCUCCGAACGUGUCACGU-3′; antisense, 5′-ACGUGACACGUUCGGAGAA-3′) were also synthesized (Qiagen, Chatsworth, CA), and served as negative controls.

Techniques: Control

Journal: Human Gene Therapy

Article Title: Small Interfering RNA Targeting Heme Oxygenase-1 (HO-1) Reinforces Liver Apoptosis Induced by Ischemia-Reperfusion Injury in Mice: HO-1 Is Necessary for Cytoprotection

doi: 10.1089/hum.2009.049

Figure Lengend Snippet: Representative histological findings in mouse liver after 90 min of warm ischemia followed by 6 hr of reperfusion in mice treated with (A) HO-1 siRNA (Suzuki score, 3.3 ± 0.5); (B) nonspecific scrambled control siRNA (score, 1.8 ± 0.7); (C) Ad-HO-1 (score, 1.2 ± 0.8); (D) Ad-β-gal (score, 3.2 ± 0.4); Results are representative of four to six mice per group; original magnification, × 200. Color images available online at www.liebertonline.com/hum.

Article Snippet: The murine nonspecific siRNA scrambled duplex (sense, 5′-GCGCGCUUUGUAGGAUUCG-3′; antisense, 5′-CGAAUCCUACAAAGCGCGC-3′) and the nonsilencing siRNA (NS siRNA: sense, 5′-UUCUCCGAACGUGUCACGU-3′; antisense, 5′-ACGUGACACGUUCGGAGAA-3′) were also synthesized (Qiagen, Chatsworth, CA), and served as negative controls.

Techniques: Control

Journal: Human Gene Therapy

Article Title: Small Interfering RNA Targeting Heme Oxygenase-1 (HO-1) Reinforces Liver Apoptosis Induced by Ischemia-Reperfusion Injury in Mice: HO-1 Is Necessary for Cytoprotection

doi: 10.1089/hum.2009.049

Figure Lengend Snippet: Intragraft neutrophil accumulation at 6 hr of reperfusion after 90 min of warm ischemia, as analyzed by MPO enzymatic activity (U/g) in ischemic lobes. Note: MPO activity was significantly increased in the HO-1 siRNA group, as compared with those given nonspecific siRNA (*p < 0.05) or Ad-HO-1 (*p < 0.05). Administration of Ad-HO-1 reduced MPO activity, as compared with the Ad-β-gal (**p < 0.005) group. Data represent four to six animals per group. Means and SD are shown.

Article Snippet: The murine nonspecific siRNA scrambled duplex (sense, 5′-GCGCGCUUUGUAGGAUUCG-3′; antisense, 5′-CGAAUCCUACAAAGCGCGC-3′) and the nonsilencing siRNA (NS siRNA: sense, 5′-UUCUCCGAACGUGUCACGU-3′; antisense, 5′-ACGUGACACGUUCGGAGAA-3′) were also synthesized (Qiagen, Chatsworth, CA), and served as negative controls.

Techniques: Activity Assay

Journal: Human Gene Therapy

Article Title: Small Interfering RNA Targeting Heme Oxygenase-1 (HO-1) Reinforces Liver Apoptosis Induced by Ischemia-Reperfusion Injury in Mice: HO-1 Is Necessary for Cytoprotection

doi: 10.1089/hum.2009.049

Figure Lengend Snippet: TUNEL-assisted detection of apoptosis in mouse liver after 90 min of warm ischemia followed by 6 hr of reperfusion. Note the dense infiltration by apoptotic cells in ischemic liver tissue in mice treated with HO-1 siRNA (A) and Ad-β-gal (D), as compared with nonspecific siRNA treatment (B; p < 0.0001). The Ad-HO-1 gene transfer group showed a decreased frequency of apoptotic cells (C; p < 0.0005) as compared with HO-1 siRNA or Ad-β-gal (D). The results were scored semiquantitatively by averaging the number of apoptotic cells (mean ± SD) per field at × 200 magnification. A minimum of six fields was evaluated per sample. Results shown are representative of three experiments. Color images available online at www.liebertonline.com/hum.

Article Snippet: The murine nonspecific siRNA scrambled duplex (sense, 5′-GCGCGCUUUGUAGGAUUCG-3′; antisense, 5′-CGAAUCCUACAAAGCGCGC-3′) and the nonsilencing siRNA (NS siRNA: sense, 5′-UUCUCCGAACGUGUCACGU-3′; antisense, 5′-ACGUGACACGUUCGGAGAA-3′) were also synthesized (Qiagen, Chatsworth, CA), and served as negative controls.

Techniques: TUNEL Assay

Journal: Human Gene Therapy

Article Title: Small Interfering RNA Targeting Heme Oxygenase-1 (HO-1) Reinforces Liver Apoptosis Induced by Ischemia-Reperfusion Injury in Mice: HO-1 Is Necessary for Cytoprotection

doi: 10.1089/hum.2009.049

Figure Lengend Snippet: (A) Activity of caspase-3 in IRI. Caspase-3 activity was markedly increased in mice treated with HO-1 siRNA, compared with those in control siRNA groups (*p < 0.001). In contrast, overexpression of HO-1 by Ad-HO-1 decreased caspase-3 activity, as compared with that of HO-1 siRNA (**p < 0.0001) or Ad-β-gal (*p < 0.001). (B) Activity of caspase-3 in Ad-HO-1-transfected YPEN-1 cells. HO-1 siRNA increased etoposide-induced caspase-3 activity, as compared with nonspecific control siRNA (**p < 0.001). In contrast, HO-1 overexpression decreased caspase-3 activity, compared with that of HO-1 siRNA (*p < 0.005) or Ad-β-gal (*p <0.005). Data shown are representative of three separate experiments. Means and SD are shown.

Article Snippet: The murine nonspecific siRNA scrambled duplex (sense, 5′-GCGCGCUUUGUAGGAUUCG-3′; antisense, 5′-CGAAUCCUACAAAGCGCGC-3′) and the nonsilencing siRNA (NS siRNA: sense, 5′-UUCUCCGAACGUGUCACGU-3′; antisense, 5′-ACGUGACACGUUCGGAGAA-3′) were also synthesized (Qiagen, Chatsworth, CA), and served as negative controls.

Techniques: Activity Assay, Control, Over Expression, Transfection

Journal: Human Gene Therapy

Article Title: Small Interfering RNA Targeting Heme Oxygenase-1 (HO-1) Reinforces Liver Apoptosis Induced by Ischemia-Reperfusion Injury in Mice: HO-1 Is Necessary for Cytoprotection

doi: 10.1089/hum.2009.049

Figure Lengend Snippet: Western blot analysis of HO-1, caspase-3, Bcl-2, and Bcl-xL gene products in hepatic lobes 6 hr after 90 min of warm ischemia. Lane 1, sham; lane 2, HO-1 siRNA; lane 3, nonspecific siRNA; lane 4, Ad-HO-1; lane 5, Ad-β-gal. Note the selectively decreased expression of HO-1 and Bcl-2/Bcl-xL, and markedly increased caspase-3, in mice treated with HO-1 siRNA as compared with those conditioned with nonspecific siRNA or Ad-HO-1. In contrast, Ad-HO-1 increased the expression of HO-1 and Bcl-2/Bcl-xL, and decreased that of caspase-3. Data shown are representative of three separate experiments.

Article Snippet: The murine nonspecific siRNA scrambled duplex (sense, 5′-GCGCGCUUUGUAGGAUUCG-3′; antisense, 5′-CGAAUCCUACAAAGCGCGC-3′) and the nonsilencing siRNA (NS siRNA: sense, 5′-UUCUCCGAACGUGUCACGU-3′; antisense, 5′-ACGUGACACGUUCGGAGAA-3′) were also synthesized (Qiagen, Chatsworth, CA), and served as negative controls.

Techniques: Western Blot, Expressing

Journal: Human Gene Therapy

Article Title: Small Interfering RNA Targeting Heme Oxygenase-1 (HO-1) Reinforces Liver Apoptosis Induced by Ischemia-Reperfusion Injury in Mice: HO-1 Is Necessary for Cytoprotection

doi: 10.1089/hum.2009.049

Figure Lengend Snippet: Quantitative real-time PCR to measure HO-1 levels in mouse liver after 90 min of warm ischemia followed by 6 hr of reperfusion. Note: Expression of mRNA encoding HO-1 was markedly depressed after treatment with HO-1 siRNA (*p < 0.05), as compared with nonspecific siRNA or Ad-β-gal. In contrast, expression of HO-1 was significantly increased in the Ad-HO-1 group, as compared with other groups (**p <0.01). Each column represents the mean ± SD (n = 3 or 4 samples per group).

Article Snippet: The murine nonspecific siRNA scrambled duplex (sense, 5′-GCGCGCUUUGUAGGAUUCG-3′; antisense, 5′-CGAAUCCUACAAAGCGCGC-3′) and the nonsilencing siRNA (NS siRNA: sense, 5′-UUCUCCGAACGUGUCACGU-3′; antisense, 5′-ACGUGACACGUUCGGAGAA-3′) were also synthesized (Qiagen, Chatsworth, CA), and served as negative controls.

Techniques: Real-time Polymerase Chain Reaction, Expressing