Figure 2. Regulation of mRNA and protein expression, and intracellular content and peptide secretion of NT by Wnt/β-catenin signaling in NET cells.

(A) Time course of upregulation of NT mRNA by LiCl. Total RNA was isolated from BON cells treated with 25 mM LiCl for the indicated times and cDNA was synthesized from 1 μg of total RNA and a high capacity cDNA reverse transcription kit (Applied Biosystems). The PCR products were visualized by 2% agarose gel (upper). β-actin was used as an internal control. Quantitative RTPCR analysis also confirmed that treatment with LiCl induced NT mRNA in BON cells (bottom). The reaction was performed using a TaqMan Gene Expression Master Mix and TaqMan probes for human NT and 18S rRNA (Applied Biosystems). Expression levels were assessed by evaluating threshold cycle (Ct) values. The relative amount of mRNA expression was calculated by the comparative ΔΔCt method (*, P < 0.05 vs. 0 h). (B) The levels of NT mRNA were checked in BON cells treated with 200 nM Wnt3a for 2 h (upper) or 5 mM iCRT3 for 24 h (middle), and in QGP-1 cells treated with 200 nM Wnt3a for 2 h (bottom). β-actin was used as an internal control. (C) The protein extracts for cell lysates were analyzed with the indicated antibodies. Western blot analysis showing induction of NT and Cyclin D1 by Wnt3a treatment in BON (upper left) and QGP-1 (upper right) cells. Western blot analysis representing suppression of NT and Cyclin D1 protein expression by 25 μM and 50 μM iCRT3 treatment in BON (bottom left) and QGP-1 (bottom right) cells, respectively. β-actin was used as a loading control. (D) NET cells were treated with vehicle or Wnt3a (200 nM) for 24 h; BON (left) and QGP-1 (right) cells were lysed and protein (50 μg) was used for NT EIA (Phoenix Pharmaceuticals) to measure intracellular NT content (*, P < 0.05 vs. vehicle). (E) The parallel medium for BON (left) and QGP-1 (right) was collected and NT peptide secretion measured by NT EIA (*, P < 0.05 vs. vehicle).