Fig 2: CS down-regulated NPAT by promoting proteasomal degradation. (A) cycloheximide C2C12 cells were treated with (CHX) (50 μg/mL) and air or air-CS mixture (the ratio of CS and air was 1:50) for the indicated times. NPAT expression in cell lysates was tested by immunoblotting analysis. Actin antibody was used as the loading control. (B) Myoblasts were cultured with air or air-CS mixture (the ratio of CS and air was 1:50), followed by incubation with DMSO (control) or the proteasome inhibitor MG132 (4 μM) for 12 h. NPAT expression in cell lysates was tested by immunoblotting analysis. Actin antibody was used as the loading control. (C) Lysates of C2C12 myoblasts treated with air and air-CS mixture (the ratio of CS and air was 1:300 or 1:50) were immunoblotted with NPAT and HiNF-P antibody. (D) FACS assay for the percentage of cells at the S phase during cell cycles in C2C12 cells treated with air or air-CS mixture (the ratio of CS and air was 1:50), transfected with control siRNA or NPAT siRNA. Values are shown as means ± SEM. N = 3. ANOVA followed by Dunnett’s post-test was applied for comparison. Significant difference from control, *p < 0.05. (E) FACS assay for the percentage of cells at the S phase during cell cycles in C2C12 cells treated with air or air-CS mixture (the ratio of CS and air was 1:50), followed by incubation with DMSO (control) or the proteasome inhibitor MG132 (4 μM) for 12 h. Values are shown as means ± SEM. N = 3. ANOVA followed by Dunnett’s post-test was applied for comparison. Significant difference from control, *p < 0.05. (F) FACS assay for the percentage of cells at the S phase during cell cycles in C2C12 cells treated with air or air-CS mixture (the ratio of CS and air was 1:50) overexpressed of FLAG or FLAG-NPAT. Values are shown as means ± SEM. N = 3. ANOVA followed by Dunnett’s post-test was applied for comparison. Significant difference from control, *p < 0.05. (G) CCK8 assay was performed on C2C12 myoblasts overexpressed of FLAG or FLAG-NPAT with or without CS (the ratio of CS and air was 1:50) treatment. Quantification analysis for OD value was performed. N = 3. ANOVA followed by Dunnett’s post-test was applied for comparison. Significant difference from control, *p < 0.05,**p < 0.01.