Fig. 3.

Continuous expression of NKX2-8 and PAX9 is essential to the tumor maintenance of amplified SCC cells. (A) Western blot analysis indicates the absence of Ttf1 protein expression in two lung SCC cell lines (H2170 and HCC15), as expected from the literature (21–24). Two additional SCC cell lines (EPLC-272H and CHAGO-K-1) were also shown by immunoblotting to lack the expression of Ttf1 protein (data not shown). Analysis of the large-cell lung cancer cell line (H661) was included as a positive control. Twenty micrograms of whole-cell extracts were loaded. The 14q13.3 amplicon status and tumor subtype are as indicated. (B and D) Effective shRNAs (indicated by an asterisk) in knocking down the endogenous protein expression of NKX2-8 or PAX9 were identified by immunoblotting of whole-cell extracts prepared from stable transfectants based in the amplified H2170 cells. Immunoblottings of β-actin or tubulin were performed to control for total amounts of proteins analyzed. (C and E) Stable H2170 transfectants expressing individual shRNAs of NKX2-8 or PAX9 were evaluated for tumorigenicity in athymic nude mice. Approximately 24 h before injections, the mice were gamma-irradiated at 400 rad to minimize residual immune responses. Subsequently, five million cells of individual transfectant populations were injected into a group of five athymic nude mice s.c., and these animals were observed weekly for tumor formation. Tumor take rate was calculated as of the last weekly tumor measurement, and the averaged tumor size (y axis) was plotted against days after injection (x axis).