FIGURE 3:

Down-regulation of hASH1 reduces the expression of p35 and Cdk5 in H727 lung cancer cells. (A) Relative expression of hASH1 mRNA by qRT-PCR in human lung cancer cell lines and bronchial epithelial BEAS-2B cells. The H727 lung cancer cell line was positive, which is consistent with the fact that it is derived from a pulmonary carcinoid. Carcinoids are well-differentiated NE cancers that express hASH1. The mRNA levels were normalized with that of the human lung adenocarcinoma A549 cell line designated as “1.” (B) Silencing hASH1 mRNA expression by shRNA in H727. By qRT-PCR, hASH1 expression was seen to be reduced more than 2.5-fold when compared with the expression by H727 cells transfected by scrambled RNA (con; p < 0.002). (C) Protein lysates from hASH1 shRNA transfected or scrambled vector–transfected cells were subjected to Western blotting. (D–E) Quantification of p35 and Cdk5 Western blot in (D) by densitometry (E; bar graph). shRNA-transfected H727 cells showed a significant reduction in p35 and Cdk5 expression compared with scrambled vector transfected cells (p < 0.002 and p < 0.05, respectively).