Fig. 4

Flow cytometry analysis of cell cycle and proliferation in HEK293 and 2D12 control and heat-shocked cells. (A) Cell cycle analysis of HEK293 grown at 37 ºC (control) and 24 h after treatment (heath shock). The percentage of cells in G1, G2, and S phases was measured as indicated in the boxes. The percentage of dead cells -D- and polyploid cells -P- was also calculated. The comparative of these populations (%) is shown in a bar diagram. (B) Same analysis in 2D12 cells. (C) Analysis of cell proliferation in HEK293 and 2D12 control and treated cells. Flow cytometry histograms of control unlabeled cells (black line) and heat shock treated cells 24 h after treatment (red line). On the right, diagram of the percentage of these populations indicated as the average of seven different replicates. (D) Flow cytometry analysis of cell proliferation in 2D12 cells transfected with the FLAG empty vector (central panel) or with the FLAG-NOA36 construct (right panel) 24 h after heat shock. The quadrants represents the following cell populations: LL: non-transfected/non-proliferating cells; UL: non-transfected/proliferating cells; LR: transfected/non-proliferating cells; UR: transfected/proliferating cells. The average percentage in three different experiments for proliferating cells in the control (empty vector, UL + URx100/(UL + LL + UL + UR) in the central panel), the FLAG-NOA36 untransfected cells (ULx100/(UL + LL) in the right panel), and the FLAG-NOA36 transfected cells (URx100/(UR + LR) in the right panel) in three different experiments are shown in the diagram. Left panel, unlabeled cells analysis with the same settings as a negative labeling control. Plots are graphed with the average and standard deviation (SD) with n = 3 in (A) and (B), and n = 7 in (C). Asterisks denote the statistical significance differences between the groups with three P-values intervals: (*) 0.01 < p < 0.05; (**) 0.001 < p < 0.01 and (***) p < 0.001 using Student’s t-test analysis