Phosphorylated Akt triggers a network that positively regulates G1/S cell cycle progression through inactivation of Gsk3�� [49], direct phosphorylation of ��-catenin at Ser552, which enhances ��-catenin nuclear accumulation [50] and increased ��-catenin-TCF/Lef selleck chem Bosutinib transcriptional activity [49]. Therefore, we investigated whether the expression of Akt and its downstream effectors was affected by NEFH down-regulation. Increased Gsk3�� phosphorylation and decreased total Gsk3�� level were observed in N12 and N20 compared to C2 cells (Fig. 3a, left). The slight increase of total Akt was due to increased Akt1 and Akt2 in N12 and N20 cells, respectively (Fig. S3a�CS3c). Figure 3 NEFH-knockdown stimulates ��-catenin-TCF/Lef signaling.

Both Gsk3�� and casein kinase-1 (CK1) phosphorylate ��-catenin at Ser33/Ser37/Thr41 and at Thr41/Ser45, respectively, leading to the degradation of ��-catenin through the ubiquitin pathway [51], [52]. In contrast, protein kinase A (PKA) phosphorylates ��-catenin at Ser675 to stabilize ��-catenin, resulting in increased cytoplasmic and nuclear ��-catenin levels [53]. In NEFH down-regulated cells, phosphorylation of ��-catenin at Ser33/Ser37/Thr41 and at Thr41/Ser45 decreased with a concomitant increase of total ��-catenin. In contrast, phosphorylation of ��-catenin at Ser675 increased in N12 and N20 cells. Activation of the Akt oncogene is in part responsible for the bioenergetic shift to dependence on glycolytic metabolism characteristic of most malignant cells [54]. Akt also plays a vital role in the maintenance of mitochondrial membrane integrity to prevent the induction of apoptosis [55], [56].

We thus further elucidated expression level of proteins that play roles in the glycolytic pathway and in mitochondrial function in NEFH-diminished cells. M2-type pyruvate kinase (PK-M2) is a key glycolytic enzyme, and pyruvate dehydrogenase (PDH) is a TCA cycle enzyme converting pyruvate to acetyl-CoA in mitochondria. PK-M2 and PDH are two of the enzymes that play a key role in increasing glycolysis and lactate production. The protein level of PK-M2 increased in N12 and N20 compared to C2 cells, whereas PDH decreased (Fig. 3a, right). A slight decrease of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was also observed in N12 and N20 cells, but little difference was observed in the expression of lactate dehydrogenase-A (LDHA), Hsp60, a mitochondrial matrix protein, or mitochondrial H+-ATP synthase (��-F1-ATPase). Interestingly, c-Myc expression, which increases mitochondrial biogenesis and cellular respiration [57], [58], was down-regulated after decreasing NEFH expression. Similar results were observed in Drug_discovery HEK293 cells transfected with NEFH siRNA (Fig. S3d).