Figure 8. Cdc42-overexpression promotes the activation of NFκB, induces increased autophagy and glycolytic metabolism. (A) GTPases are strong activators of the transcription factor NFκB. As shown in figure, note that immunoblot analysis of control or SMA-, Rac1- and cdc42- overexpressing fibroblasts reveals that p-NFκB protein levels are significantly increased, exclusively in Cdc42 fibroblasts, as compared with control fibroblasts. (B) To validate that Cdc42 induces an autophagic program, cells were subjected to immunoblot analysis using several autophagy markers. β-actin was used as an equal loading control. Note that Cdc42 increases the expression of the autophagy markers, such as Beclin-1, BNIP3, LAMP-1 and Cathepsin B (37kDA). (C) Cdc42-overexpressing fibroblasts display a predominantly glycolytic metabolism, as demonstrated by increased L-Lactate production, under hypoxic conditions. Cells cultured for 48 h under hypoxic conditions (0.5% O₂) were treated with or without metformin (1 mM), and the results are expressed as ratio between treated vs. untreated cells. (D) The shift toward a predominantly glycolytic metabolism is also demonstrated by decreased mitochondrial activity, as visualized using MitoTracker. Note that Cdc42 significantly decreases mitochondrial activity, as compared with vector alone control or SMA overexpressing fibroblasts. MitoTracker (red); nuclei/DAPI (blue). Original magnification, 60x.