Research Paper Volume 6, Issue 9 pp 755—769

Figure 5. Cell cycle dynamics is partially rescued by rapamycin in MADA fibroblasts. (A) Proliferation rate of control or MADA cells left untreated or treated with rapamycin. 1,45 × 10⁵ cells were plated at T0. After the treatment (T4), living cells were counted with trypan blue by a Neubauer camera. (B) Cell cycle analysis of cells left untreated or treated with rapamycin. The acquired FACS data were analyzed by ModFit LT software (Verity Software House, Inc.). Percentage of cells in cell cycle phases (G1, S, G2) is reported in the boxed areas within each panel. Ctrl, control. (C) Relative percentage of cells in each cell cycle phase before and after rapamycin treatment (rapamycin). (D) BrdU was incorporated into living fibroblasts for 4 hours and cells were fixed and subjected to IF staining using anti-BrdU antibody. The percentage of positive nuclei is indicated in the graph as mean value of three different counts obtained in different experiments. Data are means of three different counts +/− standard deviation of the mean. (E) Percentage of PCNA-positive MADA nuclei showing diverse staining patterns (S-phase, G1/G2 pattern). Subconfluent MADA fibroblasts left untreated (untreated) or treated with rapamycin (rapamycin) were immunolabeled for PCNA using anti-PCNA PC10 antibody. The number of nuclei with S-phase or G1/G2 staining pattern or negative for PCNA labeling was determined by counting 200 nuclei per sample.