Figure 7. Quantitative RT-PCR analysis of SK3 mRNA expression and measurements of apamin-sensitive apical membrane current in immortalized PEG cells.

A, amplification plots showing SK3 mRNA expression in confluent PEG cell monolayers under serum-free and oestrogen (E2, 20 nm, 4 days)-stimulated conditions (n = 3 for each condition). CT values for SK3 were 26.62 ± 0.04 and 24.96 ± 0.09 for serum-free and E2-treated conditions, respectively (QRT-PCR reaction efficiency = 87% based on the slope of the linear portion of the amplification plot). B, temperature dissociation plot for serum-free and oestradiol-treated samples used for generating the amplification plot shown in A (n = 3 for each condition). Note that a single peak was detected indicating that a unique PCR product was obtained. C, comparison of maximum UTP-stimulated, apamin-sensitive (100 nm), apical membrane currents of monolayers maintained for 4 days under serum-free conditions or in the presence of 20 nm oestradiol-17β. Monolayers treated with oestradiol exhibited a 2.5-fold greater apamin-sensitive inward current following stimulation with UTP (10 μm).