Figure 1.
Infection of adenovirus atrogin-1 and microarray analysis.
A. Neonatal rat cardiomyocytes were infected with adenovirus green fluorescent protein (GFP) control (Ad-GFP) or atrogin-1 (Ad-atrogin-1). The infection efficiency was visualized for GFP 24 hours later using fluorescence microscopy (Magnification, ×400). B. The levels of atrogin-1 protein were determined by Western blot analysis with anti-atrogin-1 antibody, using β-actin as the internal control. Quantitative analysis of protein bands was shown (n = 3). C. Hierarchical clustering depicting expression profiles of differentially expressed genes in Ad-atrogin-1 (A1 and A2) and Ad-control (G1 and G2) groups. Data from individual sample are shown. A subset of genes displays significant expression changes at ≥2-fold or ≤−2-fold. Gene expression levels are shown as color variations (red: up-regulated expression; green: down-regulated expression).
Figure 2.
Scatter plot of relative changes in gene expression as determined by microarray analysis and by qRT-PCR.
Eight genes were analyzed: IL-6, DKK2, Calr, Cxcl6, Cxcl1, Axin2, IL-1r1, and Cadm1. Each symbol represents the fold change of the respective gene in ad-atrogin-1 group over Ad-GFP control.
Figure 3.
qRT-PCR analysis of microarray data.
Neonatal rat cardiomyocytes were infected with Ad-atrogin-1 or Ad-GFP (A), Ad-siRNA-atrogin-1 or Ad-siRNA-control (B) for 24 hours. Total RNA was extracted and qRT-PCR analysis was performed in triplicate using specific oligonucleotides primers. The differences in gene expression levels were statistically significant. Data represent the mean±SEM (n = 3 per group). #P<0.05; *P<0.001 vs. Ad-GFP control.
Table 1.
GO:0008219 cell apoptosis and death.
Table 2.
GO:0008283 cell proliferation.
Table 3.
Metabolic pathways.
Table 4.
GO:0048468 cell development and hypertrophy.
Table 5.
GO:0006950 response to inflammation.
Figure 4.
Effects of atrogin-1 overexpression on cardiomyocyte apoptosis and hypertrophy.
Neonatal rat cardiomyocytes were infected with Ad-GFP or Ad-atrogin-1-GFP for 24 h and then treated with LPS (1 µg/ml) for additional 24 hours. A. Apoptosis was detected and quantified using TUNEL assay (red), and nuclei were counterstained with DAPI (blue). A representative field is shown for each condition (top panels), Magnification, ×400. Quantitative analysis of TUNEL-positive cells from three independent experiments (bottom panels). B. The cells were fixed and stained with anti-α-actinin antibody followed by Alexa Fluor 568-conjugated goat anti-mouse IgG (red), and nuclei were stained with DAPI (blue). A representative field is shown for each condition (top panels), Magnification, ×200. Quantitative analysis of cell surface area (a minimum of 100 randomly chosen cells measured in each group) (bottom panels). Data represent the mean ± SEM (n = 3). #P<0.05, &P<0.01 vs. Ad-GFP; *P<0.05; $P<0.01 vs. Ad-GFP+LPS. C. The qRT-PCR analysis of ANF, Myh6 and serca2 mRNA expression was performed in triplicate using specific oligonucleotides primers. D and E. Neonatal rat cardiomyocytes were infected with Ad-siRNA-atrogin-1 or Ad-siRNA-control for 24 hours. Analysis of apoptosis and cell surface area were performed as in A and B. Data represent the mean ± SEM (n = 3). #P<0.05, &P<0.01 vs. siRNA-control; *P<0.05 vs. siRNA-control +LPS.
Figure 5.
Effect of atrogin-1 on inflammation.
A. Neonatal rat cardiomyocytes were infected with Ad-GFP or Ad-atrogin-1-GFP for 24 h and then treated with LPS (1 µg/ml) for additional 24 hours. The qRT-PCR analysis of gene expression was performed in triplicate using specific oligonucleotides primers. Data represent the mean ± SEM. #P<0.05, &P<0.01, $P<0.05 vs. Ad-GFP; *P<0.01 vs. Ad-GFP+LPS. B. Neonatal rat cardiomyocytes were infected with Ad-siRNA-control or Ad-siRNA-atrogin-1 for 24 h and then treated with LPS (1 µg/ml) for additional 24 hours. The qRT-PCR analysis of gene expression was performed as in A. Data represent the mean ± SEM. #P<0.05, &P<0.01, $P<0.05 vs. siRNA-control; *P<0.01 vs. Ad-siRNA+LPS.
Figure 6.
Effects of atrogin-1 on MAPK and NF-κB signaling pathways.
A. Neonatal rat cardiomyocytes were infected with Ad-GFP or Ad-atrogin-1-GFP for 24 h and then treated with LPS (1 µg/ml). The protein levels of total and phospho-ERK1/2, JNK1/2, p38 and p65/NF-κB were detected by Western blot analysis (left panels). Quantitative analysis of relative intensity of phosphorylated proteins was shown (right panel). Data represent the mean ± SEM (n = 3). #P<0.05, &P<0.05 vs. Ad-GFP; *P<0.01 vs. Ad-GFP+LPS. B. Neonatal rat cardiomyocytes were infected with adenovirus siRNA-control or siRNA-atrogin-1 and then treated with LPS (1 µg/ml). The protein levels were detected as in A (left panels). Quantitative analysis of relative intensity of phosphorylated proteins was shown (right panel). Data represent the mean ± SEM (n = 3). #P<0.05, &P<0.05 vs. siRNA-control; *P<0.05 vs. siRNA-control+LPS.
Table 6.
Primers used for Quantitative realtime RT-PCR Analyses.