Figure 4.

GABA is required for IL-13–induced goblet cell hyperplasia in ALI cultures of NHBE cells. (A) Representative images of double staining for Ki-67 and MUC5AC in Day 21 ALI cultures of NHBE cells with and without the treatment of IL-13 and combined GABAα and GABAβ receptor blockers. Arrowheads mark MUC5AC⁺Ki-67⁺ cells. The inset shows an enlarged image of double-positive cells in IL-13–treated cultures. (B–D) MUC5AC, SPDEF (SAM pointed domain-containing Ets transcription factor), and FOXA3 gene expression in Day 21 ALI cultures of NHBE cells under different culture conditions. (E) Quantification of the percentages of MUC5AC⁺ goblet cells in Day 21 ALI cultures under different culture conditions. (F) Goblet cell proliferation in Day 21 ALI cultures under different culture conditions. Proliferation was quantified by normalizing the number of MUC5AC⁺Ki-67⁺ cells to the total MUC5AC⁺ goblet cells. More than 500 cells from each donor were counted for each condition. (G) Representative images of staining for cleaved caspase 3 in Day 21 ALI cultures with and without the treatment of IL-13 and combined GABAα and GABAβ receptor blockers. Data in B–D represent mean ± SEM of three independent experiments in triplicates for each donor, three donors in total. *P < 0.05 and **P < 0.01. Arrowheads mark caspase 3⁺ cells. Scale bars: 50 μm.