Fig 1.
Schematic description of the in vitro atrophy model and related signaling pathway investigated.
Table 1.
Primer sequences for real-time PCR.
Fig 2.
Proliferation assay of muscle-derived cells performed by time-lapse experiments at 0, 6, 12, 24, 48,72, and 96 h of incubation with HHA, LHA, and HCC at 1.6 mg/mL w/w, compared to CTR.
(A) Data shown are means±SD of four fields of view of three different wells for each sample. Proliferation experiments were repeated three times. (B) Immunofluorescence staining of myogenin in the presence of HHA, LHA, and HCC. Blue nuclei, red actin cytoskeleton, green myogenin. (C) Western blotting analyses and densitometry. Desmin and myogenin protein levels in rat muscle-derived cells treated with HHA, LHA, and HCC for 24 h. Specific bands corresponding to the proteins of interest are measured using commercially available software (Image J software). Densitometric analyses of specific bands were obtained for both the proteins, normalized to actin expression. Data shown are average of duplicates and means ± SD. The statistical significance was analyzed through the students’t-test (*p<0.01).
Fig 3.
(A) Pre-treatment with H2O2 (50 μM) for 30 minutes and post -treatment with HHA, LHA and HCC (1.6 mg/mL w/w) for 24-48-72 hours (Repair function). (B) Pre-treatment with HHA, LHA and HCC (1.6 mg/mL w/w) for 24-48-72 hours and post-treatment with H2O2 (50 μM) for 30 minutes (Protective function). Data shown are average of triplicates and means ± SD. The statistical significance was analyzed through one-way ANOVA and Tukey post hoc test for comparing a family of 5 estimates: # p<0.05 or less vs untreated-cells (CTR); ° p< 0.05 or less vs H2O2, § p<0.05 or less vs HHA;* p< 0.05 or less vs HCC.
Fig 4.
Western blotting analyses and densitometry of SOD-2.
The upper bands show the expression of the actin housekeeping protein, while the lower bands indicate the SOD-2 expression. The histograms indicate the densitometric normalized analysis on actin band for each of the cell extracts (control and treatments). (A) Pre-treatment with H2O2 (50 μM) for 30 minutes and post -treatment with HHA, LHA and HCC (1.6 mg/mL w/w) for 24-48-72 hours (Repair function). (B) Pre-treatment with HHA, LHA and HCC (1.6 mg/mL w/w) for 24-48-72 hours and post-treatment with H2O2 (50 μM) for 30 minutes (Protective function). Data shown are average of duplicates and means ± SD. The statistical significance was analyzed through the students’t-test (*p<0.01) respect to H2O2 treated cells.
Fig 5.
(A) Quantitative Real-Time PCR of atrogenes. RNA expression was determined by quantitative real-time PCR on rat muscle-derived cells treated with TNF-α along HHA, LHA, HCC at 1.6 mg/mL w/w for 24 h. Data shown as average of triplicates and each column represents the mean and error bars indicating the standard deviation. The statistical significance was analyzed through one-way ANOVA and Tukey post hoc test for comparing a family of 5 estimates: # p<0.05 or less vs untreated-cells (CTR); ° p< 0.05 or less vs TNF-α, § p<0.05 or less vs HHA;* p< 0.05 or less vs HCC. Western blotting analyses of (B) NF-kB and FoxO3a transcription factors and (C) atrogenes were performed and normalized to Actin housekeeping protein. Data shown are average of duplicates and means ± SD. The statistical significance was analyzed through the students’t-test (*p<0.01) respect to TNF-α treated cells.
Fig 6.
Desmin and myogenin were analyzed at transcriptional and protein levels in rat muscle-derived cells treated for 24 h with TNF-α and HHA, LHA, HCC at 1.6 mg/mL w/w, each.
(A) qRT-PCR was conducted to analyze the gene expression levels. The statistical significance was analyzed through one-way ANOVA and Tukey post hoc test for comparing a family of 5 estimates: # p<0.05 or less vs untreated-cells (CTR); ° p< 0.05 or less vs TNF-α, § p<0.05 or less vs HHA;* p< 0.05 or less vs HCC. (B) Western blotting analyses of specifics bands obtained for both protein expressions are normalized to actin expression. Data shown are average of duplicates and means ± SD. The statistical significance was analyzed through the students’t-test (*p<0.01) respect to TNF-α treated cells.