Research Paper Volume 12, Issue 6 pp 5195—5208

Figure 5. Methylene Blue (MB) and RAD001 reduce cellular ROS and RAD001 activated catalase activity in HGPS cells. Normal and HGPS fibroblasts were treated with 100nM Methylene Blue and RAD001 for 2 weeks. (A, B) The relative fold change of ROS activity was measured by DCFDA flow cytometry analysis. One-way ANOVA followed by Dunnett's multiple comparisons test was used to compare the effect of Methylene Blue and RAD001 treatment with the control group (Vehicle). *, p < 0.05, **, p < 0.01, ***, p < 0.001. (C) Relative mRNA expression of catalase was detected by quantitative RT-PCR analysis. Two-way ANOVA followed by Dunnett's multiple comparisons test was used to compare the mRNA expression of Methylene Blue and RAD001 treated cells with the control group within each block (normal and HGPS). n.s., not significant, **, p < 0.01. (D) Catalase expression level was detected by Western blot analysis (cell passage number = 19). (E) Normalized catalase activity. Two-way ANOVA followed by Dunnett's multiple comparisons test was used to compare the catalase activity of Methylene Blue and RAD001 treated cells with the control group within each block (normal and HGPS). n.s., not significant, **, p < 0.01. All experiments were repeated at least three times and representative data were shown as indicated.