Fig 2: Succinate triggers ATP release from PNECs to elicit SMG contraction.A) Representative images of Fura-2 ratiometric Ca2+ imaging in DMS454 cells before and after succinate treatment. Arrowheads highlight the cell’s response to succinate.B) Summary of ratiometric Ca2+ imaging (R/R0, R = 340/380) in DMS454 and DMS53 cells that were treated with succinate or methyl succinate. Data are mean ± SEM.C) Relative SMG area 3 minutes after CGRP (0.5 µM), 5-HT (40 µM), ATP (1 mM), and carbachol (10 µM) treatment. Each data point is the mean of 3 SMGs from a pig; p = 0.0012, one-way ANOVA.D) Representative smFISH images of SMG PNECs (KRT20 and ASCL1+) expressing SLC17A9 (VNUT).E) qRT-PCR of SUCNR1 and SLC17A9 in DMS53 and DMS454 cells.F) Representative immunofluorescent images of VNUT (green) and CGRP (red) in DMS454 cells. Enlarged image highlights the colocalization of VNUT and CGRP.G) ATP release from DMS454 cells treated with methyl succinate (5 mM), succinate (5 mM), or ionomycin (2 µM). Each data point is the mean of replicates from an independent experiment. Lines connect the same experiment across treatment groups; p = 0.006, paired Student’s t test. See also Figure S6.

Fig 3: Porcine and human PNECs express SUCNR1.A) G protein-coupled receptors in SMG scRNA-seq data (Yu et al., 2022). Genes are ranked by false discovery rate (FDR) from the lowest to the highest. Ser, serous cell; Myoep, myoepithelial cell; Muc, mucous cell. See also Table S1.B-D) Characterization of SUCNR1 expression using smFISH in porcine PNECs. B is representative smFISH images of SUCNR1, ASCL1, and POU2F3 in SMGs and airway surface epithelia (ASE). C is the percentage of cells that are positive for SUCNR1 for PNECs (224 cells) and tuft cells (88 cells). n = 4 pigs (2WT, 2CF); p < 0.0001, paired Student’s t test. D is normalized SUCNR1 level (smFISH counts) in WT and CF PNECs (224 cells). P value was calculated based on individual SMGs; p > 0.05 is considered as not significant (n.s), unpaired Student’s t test.E) Apical distribution of SUCNR1 in airway surface epithelia. Newborn pig trachea was infected with lentivirus carrying CMV promoter driven Myc-tagged SUCNR1 cDNA. After one week of ex vivo culture, tissues were sectioned and stained with anti-Myc antibody. Dashed line highlights airway surface, arrowheads point to cells with apical Myc tag staining.F) Representative immunofluorescent images of neuron fibers (SYP+, green) and PNECs (KRT20+, gray) in porcine SMGs (EPCAM+, red). Arrowhead highlights the proximity of PNECs and neurons.G) Uniform manifold approximation and projection (UMAP) of pig, human, and mouse airway rare cell types grouped by cell-type (left) and organism (right).H) Expression of PNEC markers (ASCL1, INSM1) in UMAP of pig, human, and mouse airway rare cell types.I) Expression of SUCNR1 in the PNEC cluster of human, pig, and mouse airway scRNA-seq data.J) Percentage of SUCNR1+ cells in PNECs and tuft cells (tuft1 plus tuft2) in human, pig, and mouse airway scRNA-seq datasets. Data points represent biological samples from the original scRNA-seq datasets. P values are shown in graph, unpaired Student’s t test.K) Comparison of PNEC markers between human and pig vs. mouse. Marker genes for both human and pig PNECs are shown. LogFC, fold change at natural log scale. See also Figure S3.