Figure 1. The effect of increased extracellular and intracellular calcium on β-defensin mRNA expression. (A) RT-PCR analysis. HGECs were incubated in various calcium concentrations (0.03, 0.06, 0.15, 0.60, or 1.20 mM) overnight. Total RNA was isolated and RT-PCR was conducted as described in MATERIALS & METHODS. The sizes of the amplified products for hBD-1, -2, -3, IL-8, and GAPDH are indicated and were as predicted. Note that hBD-2 and IL-8 mRNA were induced by increased extracellular calcium concentration in a dose-dependent manner, while hBD-1 and hBD-3 mRNA expression was not affected. A -RT sample was a negative control where enzyme was omitted. The data shown are representative of 5 separate experiments. (B) Densitometric analysis of hBD-2 expression as a function of calcium concentration. The relative ratios of hBD-2 to GAPDH were determined as described in MATERIALS & METHODS. The y axis represents the relative hBD-2 mRNA expression for the different calcium concentrations as shown in panel A. The results are represented as means plus standard deviations of 5 separate experiments. (C) RT-PCR analysis. HGECs were stimulated with various thapsigargin concentrations (0, 1, 10, 100, or 1000 nM) overnight. Note that hBD-2 and IL-8 mRNA were induced by thapsigargin in a dose-dependent fashion, whereas hBD-1 and hBD-3 mRNA expression was not affected. The data shown are representative of 4 separate experiments. (D) Densitometric analysis of hBD-2 expression as a function of thapsigargin concentration as shown in panel C. The results are represented as means plus standard deviations of 4 separate experiments.