Research Paper Volume 6, Issue 9 pp 755—769

Figure 3. LAP2alpha and Oct-1 localization are affected in MADA and rescued by rapamycin. (A) Control, RD and MADA cells left untreated (untreated) or after rapamycin treatment (rapamycin) were stained for LAP2alpha. Levels of LAP2alpha fluorescence intensity and the percentage of nuclei with LAP2alpha mislocalized in nuclear clusters are reported in the upper and lower graph respectively as mean values +/− standard deviation. (B) Control, RD and MADA cells left untreated (untreated) or after rapamycin treatment (rapamycin) were stained for Oct-1. Arrows indicate Oct-1 nuclear foci, arrowheads indicate recovered Oct-1 localization in the nucleoplasm. The pseudo-coloring of Oct-1 pictures obtained by using the Photoshop 7 color mapping function reveals the accumulation of Oct-1 in nuclear foci and at the periphery and recovery by rapamycin treatment in MADA. The intensity of fluorescence is represented on a pseudocolor scale (palette bar). The percentage of nuclei with Oct-1 mislocalized in nuclear clusters is reported in the graph as mean values +/− standard deviation. (C) Proximity Ligation Assay (PLA) between prelamin A and Oct-1. Control and MADA fibroblasts left untreated or treated with rapamycin, were labeled with anti-Oct-1 and anti-prelamin A (SC - 6214) antibodies and probed with Duolink (Sigma) detection reagents according to the manufacturer. Nuclei were counterstained with DAPI. The PLA signals (in red) were counted with the Duolink ImageTool software and the average number of spots in the nucleus per cell (200 cells per sample were counted) is presented in the graph. Statistically significant differences (p<0.05) are indicated by asterisks.