Research Paper Volume 6, Issue 9 pp 755—769

Figure 2. Prelamin A degradation in RD and MADA cells. (A) Cellular lysates from control, RD or MADA fibroblasts were subjected to Western blot analysis to test prelamin A, lamin A, lamin C and LC3B2 levels under different experimental conditions. Actin was labeled as a loading control. Cultured fibroblasts were left untreated or treated with rapamycin and/or chloroquine. Densitometric analysis of lamin A, lamin C and prelamin A immunoblotted bands is shown (dark red bars highlight untreated samples). Statistically significant differences (p<0.05), measured by the Mann-Whitney test, are indicated by asterisks. Prelamin A was detected by anti-prelamin A antibody (Santa Cruz Sc-6214), lamin A/C by anti-lamin A/C antibody (Santa Cruz Sc-6215). (B) Prelamin A immunofluorescence staining performed using anti-prelamin A 1188-1 antibody, which labels non-farnesylated prelamin A, was detected using a TRITC-conjugated secondary antibody (red). (C) Prelamin A immunofluorescence staining performed using anti-prelamin A 1188-2 antibody, which labels farnesylated prelamin A, was detected using a FITC-conjugated secondary antibody (green). Control, RD or MADA fibroblasts were left untreated (−) or treated with rapamycin (+) for 4 days. Bar, 10 μm. Statistical evaluation of non-farnesylated and farnesylated prelamin A-labeled nuclei is reported in the graphs in (B) and (C), respectively. 200 nuclei per sample were counted. Western blots showing non farnesylated and farnesylated prelamin A on the same treated or untreated cultures are also shown in (B) and (C), respectively. Densitometric values of prelamin A bands in rapamycin-treated samples are reported below each immunoblotted band as percentage of the intensity detected in the corresponding untreated sample. (D) Lamin A/C staining of control, RD and MADA fibroblasts showing misshapen nuclei in laminopathic cells left untreated (untreated) or subjected to 4 day rapamycin treatment (rapamycin). The quantitative analysis is reported in the graph. Statistically significant differences (p<0.05) relative to untreated corresponding samples were calculated using the Mann-Whitney non-parametric test and are indicated by asterisks.