Effect of IGF-1 on p65 nuclear localization and NF-κB pathway activation in Panc02 cells.
A, Representative fluorescent images of p65 localization at various time points following IGF-1 treatment (400 ng/mL) using 300 nM DAPI alone, a p65 antibody alone (p65), or p65 antibody counterstained with DAPI (merged). Data representative of 4 separate assays. B, ELISA of p65 DNA binding in response to 4-hour IGF-1 treatment (400 ng/mL). C, NF-κB luciferase reporter assay after 6-hour IGF-1 treatment (400 ng/mL). *denotes significant differences between SF and IGF-1. D, PCR analysis of mRNA expression of NF-κB downstream gene targets after 18-hour IGF-1 treatment. E, ELISA measurement of RANTES, LIF, and VEGF (n = 3) expression in Panc02 supernatant after 24 hours of IGF-1 treatment. *denotes significant differences between SF and IGF-1 with respect to each respective gene or protein of interest. All experiments used 400 ng/mL of IGF-1. Bar graphs represent mean ± SEM (n = 3 separate experiments performed in triplicate for B, C, and D). SF, serum-free; IGF-1, insulin-like growth factor-1; DAPI, 4′-6-diamidino-2-phenylindole; RANTES, Regulated on Activation, Normal T cell Expressed and Secreted; LIF, leukemia inhibitory factor; VEGF, vascular endothelial growth factor.