Fig. 2

Establishing chemically induced Paneth cell (PC)-enriched cultures. a Schematic of small molecule-driven differentiation of LGR5+ ISCs (C - CHIR99021, D - DAPT) and non-specific differentiation. b mRNA expression of PC (Lyz1, Defa1, Mmp7) and ISC (Lgr5) markers relative to ENR, for ENR+CV and ENR + CD at 2 (D2), 4 (D4), and 6 days (D6) (n = 3 biological replicates; two-way ANOVA with multiple comparison test vs. ENR; ** adj. p < 0.01, *** adj. p < 0.001). c Representative confocal imaging of whole cell clusters for PC antimicrobials following 6 days in ENR+CD versus ENR and ENR+CV: stained for anti-DEFA, anti-LYZ and counterstained with DAPI and for actin (phalloidin). d High-resolution fluorescent imaging of in vivo and in vitro single cells from 6-day culture in ENR + CD shows similar morphology and antimicrobial expression: stained for DEFA and LYZ, and counterstained with DAPI and for actin (phalloidin). e Viable cell populations from ENR, ENR+CD, and ENR+CV precursor culture have distinct populations based on CD24 and LYZ content, indicative of PC maturity (n = 3 biological replicates; ENR + CV, days 4, 6, 12, n = 2 biological replicates day 8). f Volcano plot of differentially regulated proteins between 6-day (6D) ENR + CD and ENR cells shows clear enrichment in secreted and PC-associated proteins (labeled). Cut-offs are 2 standard deviations outside the mean expression level of the set and FDR < 0.05. g Rank-order log fold change of detected PC antimicrobial proteins and between 6-day ENR + CD and ENR cultures (n = 4). h Rank-order log fold change of detected secretory proteins associated with EEC and goblet lineages in ENR + CD relative to ENR cultures (n = 4)