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. 2013 Apr 3;110(16):6524–6529. doi: 10.1073/pnas.1303932110

Fig. 3.

Fig. 3.

TrkB and NCAM are downstream targets of NeuroD1 that phenocopy loss of NeuroD1. (A) XY scatter plots of 35 adenocarcinoma and squamous patient samples examining correlation between NEUROD1, NRTK2 (TrkB), and NCAM. P values and R2 values were obtained by Pearson’s test. (B) qRT-PCR analysis of NRTK2, NCAM, and NEUROD1 in lung cancer cells with stable knockdown of NeuroD1. (C) ChIP of NeuroD1 on NTRK2 promoter in cell lines expressing shNeuroD1 or shcontrol (GIPZ). One of four independent experiments in duplicate. (D) ChIP of NeuroD1 on two E boxes in the NCAM promoter with NeuroD1 consensus binding sites in HBEC3KT, three SCLCs, and a NSCLC-NE. NeuroD1 immunoprecipitation values were compared with input, and then plotted as percentage chromatin fold enrichment normalized to HBEC-3KT. (E) Soft agar assay of SCLC and NSCLC-NE lines infected with shControl or shTrkB. The average numbers of colonies after 2 wk are shown. Error bars indicate ±SD from the mean of four independent experiments in triplicate (***P < 0.001; one-way ANOVA). (F) Cell lines were infected with shControl or shNCAM and subjected to soft agar assay. The average numbers of colonies after two weeks are shown. Error bars indicate ±SD from the mean of two independent experiments in triplicate. (G) HBEC3KT and HBEC30KT cells were transfected with a plasmid encoding human TrkB, and then subjected to Transwell assay. Graph represents fold mean ± SD of four and three independent experiments, respectively (**P < 0.005, *P < 0.05; one-way ANOVA). (H) Mice were injected with 106 H1155 cells infected with shNeuroD1 or shControl. Tumors were measured until maximum tumor burden was reached (n = 10, 5 mice per group). P values were computed by linear regression (of slopes) for volume measurements. Means are ±SEM. (I) H69, H82, and H1155 were cell lines were subjected to knockdown of NeuroD1 and/or NeuroD1/TrkB. Knockdown cells were then subjected to overexpression of either NeuroD1 or TrkB. Cells were then embedded in growth factor-reduced Matrigel, for Transwell migration assays as described in Experimental Procedures.