Figure 4. MSF overexpression in fibroblasts induces a shift toward glycolytic metabolism. (A) Under hypoxic conditions, MSF fibroblasts secrete elevated levels of L-lactate, consistent with increased aerobic glycolysis. As shown in the figure, MSF fibroblasts secrete increased levels of L-lactate (~2-fold, p = 0.004; normalized for protein content; ~1.5-fold, p = 0.03; normalized for cell number), relative to control fibroblasts processed in parallel. This suggests that MSF-overexpressing fibroblasts could provide essential energetic support for adjacent tumor epithelial cells, via the secretion of L-lactate, which is a critical mitochondrial fuel for the tumor growth. (B) MSF overexpression induces a decrease in mitochondrial activity, as visualized using MitoTracker staining. Note that MSF decreases mitochondrial activity, both under normoxic condition (upper panels) and under hypoxic condition (lower panels). MitoTracker (red); nuclei/DAPI (blue). Original magnification, 60×. (C) MSF activates the Akt pathway. Control or MSF fibroblasts were subjected to immunoblot analysis with antibodies to phospho-Akt, phospho-mTOR and phospho-p70 S6 kinase. The phospho-Akt, phospho-mTOR and phospho-p70 S6 kinase immunoblots were then reprobed with Akt, mTOR and p70 S6 kinase antibodies, respectively. Note that MSF overexpression is associated with Akt pathway activation. This reaction is probably an anti-apoptotic response, to protect the fibroblasts from autophagy-induced cell death.