Fig. 3.

In vitro characterization of iSGs. a Schematic representation (top) and phase-contrast representative image of the iSG isolated from d23 aggregates (bottom). Scale bar, 300 μm. b, c A hierarchical cluster analysis (b) and principal component analysis (c) based on global gene expression examined by RNA-seq. The data are shown for iSGs on d23, mouse ESCs, and SMG at E13.5, E15.5, E18.5, and 6 weeks. The blue circle outline indicates embryonic SMGs. d Gene expression levels of pluripotent markers and embryonic and adult salivary gland-specific markers were compared. e Real-time RT-PCR verification of the RNA-seq data. Gene expression levels of salivary gland cell lineage markers in ESCs, embryonic and adult SMG, and iSGs were normalized to GAPDH and are presented as the fold change compared with the mean ± S.D. of triplicate samples. This experiment was replicated three times with similar results. f iSGs were stimulated with two concentrations of CCh (100 µM, 10 µM) or pretreated with atropine (ATR), followed by 100 µM CCh. Changes in the Fluo-4 fluorescence intensity were recorded. This experiment was replicated three times with similar results