Figure 6. Time-dependent changes in the transcriptional regulatory network in NecB-treated young and old HDF cells. (A and B) After NecB treatment of young and old HDFs seeded in a 96-well plate, the expressions of several transcription factors were investigated by measuring the fluorescence intensity every 12 h for 10 days in cells transduced with a lentiviral vector containing transcription factor binding sites. The transcriptional regulatory network was inferred by BTNET. The color of each circle in A indicates the expressional change of each transcription factor (red for up-regulation and green for down-regulation) and the arrow between circles indicates edge information (dotted line for inference and solid line for prior knowledge). The time-dependent activation of transcription factor YY1 in NecB-treated young and old HDFs was plotted in B. (C) Young and old HDFs were treated with vehicle or 10 μg/ml NecB for 24-48 h. Cell lysates were analyzed by western blotting with antibodies against YY1, Snail, Bcl-xL, Bcl2, P-STAT3, STAT3, and β-actin. (D) The band density of the immunoblot was quantitated by using Image J software and normalized to β-actin. The relative protein levels (fold) were calculated and plotted as the mean ± SEM.