Fig. 3.

Effect of CTL knockdown on choline transport and ACh secretion. CTL subtypes 1–5 and CHT1 were knocked-down by RNAi and cells processed as described in the methods. A. Isoform specific siRNAs decreased levels of CTL1, 2, 4 and 5 mRNA respectively by 50–70% compared to control siRNA-treated cells without affecting levels of other CTL’s. Knockdown for CHT1 and CTL3 are not shown since they were below the level of detection in H82 cells and were included as negative controls for effects on ACh secretion. Data are mean ± SE of at least four replicates in two separate experiments. *P < 0.05 compared to RNA levels in cells treated with control (nonsense) siRNA. B. Choline uptake in Na⁺-free buffer was significantly decreased compared to control siRNA in H82 cells by knockdown of CTL 1, 2 or 5, but not by knockdown of CTL4 (control choline uptake equaled 10.04 ± 1.66 pmol/mg/15min, choline concentration = 20 nM). Data are mean ± SE of 4 replicates in two separate experiments. *P < 0.05 compared to choline uptake in cells treated with control siRNA. C. Effect of knockdown of CTLs 1–5 and CHT1 on ACh secretion by H82 cells. Only knockdown of CTL4 significantly reduced ACh secretion CTL4 (control ACh secretion equaled 80.2 ± 29.9 pmol/0.5 million cells/24h). Knockdown of CTL3, 5 and CHT1 had no effect, while knockdown of CTL1 and 2 significantly increased levels of ACh secretion. 50 μM neostigmine added to all cultures to prevent ACh degradation. Data are mean ± SE of 6 replicates in three separate experiments. *P <0.05 compared to the cells treated with control siRNA.