Fig. 8: Loss of NHE1 activity impaired Paneth cell differentiation.

a–c Lineage tracing for Paneth cells from Lgr5⁺ ISCs without and with NHE1 inhibition. In (a), representative images showing the presence Lgr5⁺ ISC progeny expressing RFP and lysozyme (LYZ) (arrowhead) in the crypt region (CD44⁺) of day 3 control but not the EIPA-treated organoids. In (b), percent of crypt region (mean ± SD) in day 3 organoids contains RFP⁺, LYZ⁺ cells (n = 3 biologically independent experiments, two-sided Mann–Whitney test). Source data are provided as a Source Data file. In (c), quantification of the number of RFP⁺, LYZ⁺ cells in the crypt region of day 3 organoids (n = 3 biologically independent experiments, two-sided Mann–Whitney test). Source data are provided as a Source Data file. d–f lineage tracing for newly produced Paneth cells from the secretory progenitors during the development of Atoh1^CreERT2;Rosa26^tdTomato organoids in the absence and presence of EIPA. In (d), representative frames (day 1 and day 2) from the time-lapse recordings. Newly produced Paneth cells are indicated by the presence of visible dense granules (arrowhead) that are also marked by the secretory lineage marker ATOH1 (tdTomato⁺). Data from n = 3 biologically independent experiments are normalized to either (e) the number of all labeled secretory cells (tdTomato⁺), or (f) the number of non-Paneth secretory cells (tdTomato⁺, non-Paneth) on day 1. Two-sided Mann–Whitney test. Source data are provided as a Source Data file. g, h Paneth cell abundance in crypts during organoid development from day 1 to day 3. In (g), representative 3D confocal images show Paneth cells in organoid crypts at the indicated days after plating without and with EIPA. CD44, crypt region. LYZ, Paneth cell. Quantification of Paneth cell numbers (h) from conditions described (g) (n = 3 biologically independent experiments; two-sided Mann–Whitney test and two-sided student’s t test). Source data are provided as a Source Data file. All violin plots are minimum to maximum, the box shows 25th-75th percentiles, and the median is indicated as a central line. Data in plots with a statistically significant difference specified with p values, otherwise are not significantly different. All scale bars, 20 μm.