Lactate full read levels and glucose consumption were reduced by NEFH expression (Fig. 4f). In contrast, NEFH did not affect energized mitochondria (red fluorescence, J-aggregates), but increased the level of JC-1 monomers (green fluorescence); ����m decreased in NEFH-transfected cells (Fig. 4g). The population of control cells in the bottom right quadrants (LR) was 4.15%, and increased to 9.15% in NEFH-transfected cells (data not shown). Significant increases of oxygen consumption and ATP generation were observed in NEFH-transfected cells (Fig. 4h), whereas oligomycin, an inhibitor of mitochondria H+-ATPase, inhibited the NEFH-increased oxygen consumption and ATP generation. Concomitantly, a 3-fold increase of ROS level was observed (Fig. 4i).

These results indicate that NEFH regulates mitochondrial function, leading to alterations of cellular respiration and glycolysis. ��-Catenin Is Required for the Inverse Regulation between PDH and PK-M2 Gsk3�� is a key enzyme that phosphorylates ��-catenin at NH2-terminal serine threonine residues. Gsk3�� inactivation is mediated mainly by two pathways: 1) activation of growth factor receptors by EGF, platelet-derived growth factor or insulin, leading to activation of Akt, a protein kinase that phosphorylates and inactivates Gsk3��, 2) activation of the Wnt pathway leading to inhibition of Gsk3��. To investigate whether the Akt-��-catenin pathway is involved in the regulation of PK-M2 and PDH, each siRNA pool targeting PTEN, Gsk3��, ��-catenin, or a non-targeting control was transfected into C2 and/or N20 cells, and western blot analysis was performed 48 hrs after transfection.

As shown in Figure 5, more than 90% of each endogenous gene level was knocked down by the transfection. Activation of Akt with increased expression of ��-catenin and PK-M2 was observed in C2 cells with PTEN gene knockdown (Fig. 5a, left). Gsk3�� knockdown slightly increased the expression of PK-M2 in N20 cells, but not in C2 cells (Fig. 5a, right), indicating differences in knockdown efficiency of Gsk3�� or the presence of Gsk3��-independent PK-M2 regulation in C2 cells. However, PDH expression was decreased by PTEN or Gsk3�� siRNA transfection. In addition, ��-catenin knockdown reversed the increased PK-M2 expression, and reversed the decreased level of PDH in N20 cells (Fig. 5b, left). Next, we transfected the pCI-��-cat plasmid expressing wt-��-catenin or mock control into KYSE140 cells and compared expression levels of PK-M2 and PDH. Over-expression of ��-catenin increased PK-M2 but decreased PDH levels (Fig. 5b, right). These results indicate that PK-M2 and PDH are downstream effectors of the Akt-��-catenin pathway signaling in N20 cells. Figure 5 PK-M2 and AV-951 PDH are regulated through the Akt-��-catenin pathway.