2d, right). Figure 2 Down-regulation sellectchem of NEFH increases tumor growth. To examine the loss of NEFH in vivo, cells were subcutaneously injected into the flanks of 6-week-old nude mice. Tumor development was observed 10 days after injection in both flanks injected with control or either N12 (n=6) or N20 cells (n=11). A marked increase of tumor volume was observed in mice injected with NEFH-knockdown cells (Fig. 2e�Ch). Tumor volume in the mice injected with C2 cells did not change significantly for 4 weeks after injection. IHC analysis confirmed decreased expression of NEFH in N12- or N20 cells-grafted tumors (Fig. S2d). The expression of proliferating cell nuclear antigen (PCNA), a marker for cell proliferation, increased in N12 and N20 cells.

To examine the effects of increased NEFH expression on the growth of esophageal cancer cells, the KYSE140 cell line was selected due to its barely detectable basal expression of the NEFH gene (Fig. 1e). KYSE140 cells with a higher level of NEFH did not survive for longer than three weeks (data not shown), so transient transfection was performed for NEFH gene delivery. We first performed the MTT assay to compare cell growth with or without expression of NEFH. The growth in cells expressing NEFH decreased to 55% of control cells that exponentially grew for 3 days of incubation, (Fig. 2i) (P<0.05). We then performed the colony focus assay. In control cells, KYSE140 exhibited strong colony-forming ability with multiple colonies (125��11.24 colonies) (Fig. 2j). However, in pNEFH-transfected cells, a marked decrease in colony numbers was observed (55.

33��6.43 colonies, 44% of control) (P<0.001). These results indicate that forced expression of NEFH suppresses cell growth. To investigate whether NEFH had apoptotic activity, the population of live and dead cells were determined by the trypan blue exclusion assay. The population of non-viable, apoptotic cells having blue cytoplasm was less than 2% in control cells, whereas the population of non-viable cells in pNEFH-transfected cells increased to 27% (Fig. 2k). The apoptotic cell population was further assessed by flow cytometry after staining cells with Annexin V-FITC and 7-AAD, based on the cell population in the top right (late stage of apoptosis) and the bottom right quadrants (early stage of apoptosis).

A 15% increase in apoptotic cells was observed in pNEFH-transfected cells compared to control with a distribution shift toward increased apoptosis (Fig. 2l). Forced expression of NEFH caused a 2-fold increase in the caspase?3/7 activity (Fig. 2m), but did not increase the caspase-9 activity (data not shown). Western blot analysis showed increased levels of cleaved PARP (C.PARP) and BAX, but decreased level of Bcl-2 in NEFH Dacomitinib expressing cells (Fig. 2n), indicating that the mitochondria-mediated cell death pathway is activated by NEFH.