Fig 1.
Effects of hypoxia/reoxygenation on cell viability and HO-1 expression levels in H9c2 cells.
(A) H/R-induced cytotoxicity in H9c2 cells. Cell viability was measured using the CCK-8 assay. The data are presented as means ± SE (N = 5). ***p<0.001 vs. the normoxia group. (B) Real-time quantitative PCR (RT q-PCR) of HO-1 mRNA expression in H9c2 cells subjected to H/R for the indicated time relative to GAPDH expression (n = 3 wells per group). **p<0.01 vs. the normoxia group. (C) Time course of HO-1 protein expression analyzed using western blots of H9c2 cells. The expression of GAPDH was used as a loading control (n = 3 wells per group). *** p<0.001 vs. the normoxia group. H/R, Hypoxia/reoxygenation; HO-1, heme oxygenase-1.
Fig 2.
Hypoxia/reoxygenation-induced mitochondrial dysfunction and autophagy level in H9c2 Cells.
(A) Cell viability was measured using the CCK-8 assay. The data are presented means ± SE (N = 5). ***p<0.001 vs. normoxia group. ###p<0.001 vs. corresponding H/R group. (B) H/R induced HO-1, p62, and LC-3 protein expression analyzed using western blots of H9c2 cells. GAPDH was used as a loading control (n = 3 wells per group). *p<0.05 vs. corresponding normoxia group; #p<0.05, vs. corresponding H/R group. (C) Representative confocal microscopy images and quantitative analysis of autophagosomes from 15 fields (n = 3 hearts per group). Scale bar = 500 nm. **p<0.01 vs. corresponding normoxia group; ##p<0.01 vs.corresponding H/R group. H/R, Hypoxia/reoxygenation; HO-1, heme oxygenase-1. (D,E) Flow cytometry detection of changes in JC-1 fluorescence color reflects changes in the mitochondrial membrane potential and mitochondrial ROS levels. **p<0.01 vs. corresponding normoxia group; ***p<0.001 vs corresponding Normoxia group. ##p<0.01, vs corresponding H/R group. (F) The apoptosis rate of the four groups. Cell identification and detection of apoptosis. **p<0.01 vs corresponding Normoxia group; #p<0.05, vs corresponding H/R group.
Fig 3.
Effects of HO-1 overexpression on mitochondrial dysfunction and autophagy level in Lv-HO-1-H9c2 cells with H/R model.
(A) Cell viability was measured using the CCK-8 assay. The data are presented as means ± SE (N = 5). **p<0.01 vs corresponding Normoxia group; #p<0.05, vs corresponding Lv-scramble group. (B) Real-time quantitative PCR (RT q-PCR) analyses of HO-1 mRNA expression in lv-HO-1 H9c2 cells subjected to H/R relative to GAPDH expression (n = 3 wells per group). **p<0.01 vs. the normoxia group. (C) H/R-induced HO-1, p62, and LC-3 protein expression analyzed using western blots of lv-HO-1 H9c2 cells. GAPDH was used as a loading control (n = 3 wells per group). ##p<0.01 vs corresponding Lv-scramble group; ***p<0.001 vs. the corresponding normoxia group. (D) Representative confocal microscopy images and quantitative analysis of autophagosomes from 15 fields (n = 3 hearts per group). Scale bar = 500 nm. *p<0.05 vs. the corresponding normoxia group; ##p<0.01, vs corresponding Lv-scramble group. H/R, Hypoxia/reoxygenation; HO-1, heme oxygenase-1. (E,F) Flow cytometry detection of changes in JC-1 fluorescence color reflects changes in the mitochondrial membrane potential and mitochondrial ROS levels. #p<0.05, vs corresponding Lv-scramble group; **p<0.01 vs corresponding Normoxia group, #p<0.05, vs corresponding Lv-scramble group. (G) The apoptosis rate of the four groups. Cell identification and detection of apoptosis. **p<0.01 vs corresponding Normoxia group; ##p<0.01, vs corresponding Lv-scramble group.
Fig 4.
Effects of the HO-1 inhibitor ZnPP on mitochondrial dysfunction and autophagy level in H9c2 cells with H/R model.
(A) Cell viability was measured using the CCK-8 assay. The data are presented as means ± SE (N = 5). **p<0.01, vs corresponding Normoxia group. (B) Real-time quantitative PCR (RT q-PCR) of HO-1 mRNA expression from H9c2 cells subjected to the HO-1 inhibitor ZnPP relative to GAPDH expression (n = 3 wells per group). **p<0.01, vs corresponding Normoxia group. (C) H/R induced HO-1, p62, and LC-3 protein expression analyzed using western blots of H9c2 cells subjected to the HO-1 inhibitor ZnPP. GAPDH was used as a loading control (n = 3 wells per group). *p<0.05 vs corresponding Normoxia group; #p<0.05, vs corresponding H/R group. (D) Representative confocal microscopy images and quantitative analysis of autophagosomes from 15 fields (n = 3 hearts per group). Scale bar = 500 nm. **p<0.01 vs corresponding Normoxia group. (E,F) Flow cytometry detection of changes in JC-1 fluorescence color reflects changes in the mitochondrial membrane potential and mitochondrial ROS levels. **p<0.01 vs. the corresponding normoxia group. (G) The apoptosis rate of the four groups. Cell identification and detection of apoptosis. **p<0.01 vs corresponding Normoxia group.
Fig 5.
The vector map of pHBLV-CMVIE-IRES-puro.
EcoRI and BamHI are insertion sites.
Table 1.
Primer design.