Research Paper Volume 6, Issue 9 pp 755—769

Figure 4. Chromatin-associated proteins affected in MADA are rescued by rapamycin. (A) Western blot analysis of SIRT-1 in control and MADA lysates left untreated or after rapamycin treatment. The lysates were obtained in SDS buffer (insoluble) or in RIPA buffer (soluble) to evaluate the amount of SIRT-1 in different nuclear platforms; densitometric values of immunoblotted SIRT-1 bands are shown in the graph as means +/− standard deviation. Statistically significant differences (p<0.05) measured by the Mann-Whitney test are indicated by asterisks. (B) SIRT-1 staining in control or MADA cells left untreated (untreated) or subjected to rapamycin treatment (rapamycin). Non-extracted nuclei (−), nuclei subjected to detergent extraction (NP40) or to DNase treatment and high salt extraction (DNase) are shown. Nuclei were counterstained with DAPI. Scale bar, 10 μm. (C, D) Control, RD and MADA cells left untreated (untreated) or after rapamycin treatment (rapamycin) were stained for trimethyl-H3K9 (tri-H3K9) or acetyl-H3K9. Tri-H3K9 antibody labeling was revealed by FITC-conjugated secondary antibody (green), acetyl-H3K9 antibody labeling was revealed by TRITC-conjugated secondary antibody (red). (E) Tri-H3K9 and acetyl-H3K9 mean fluorescence intensity values measured by the NIS software in 200 nuclei are plotted. Statistically significant differences (p<0.05) are indicated by asterisks. Black asterisks indicate significance versus control cell cultures, red asterisks indicate significance versus the corresponding untreated sample. (F) Western blot analysis of trimethyl-H3K9 (tri-H3K9) and acetyl-H3K9 in control (ctrl) or MADA fibroblasts (MADA) before or after rapamycin treatment. Total H3 is reported as a loading control. The densitometric analysis of immunoblotted tri-H3K9 and acetyl-H3K9 bands is reported in the graph as mean values +/− standard deviation of the mean obtained in three different experiments. Statistically significant differences (p<0.05) measured by the Mann-Whitney test are indicated by asterisks.