Fig. 1: A pHi gradient develops in small intestinal organoid crypts.
From: Intracellular pH dynamics regulates intestinal stem cell lineage specification
a Schematic representation of small intestinal organoids growth. b–d pHi in the intestinal crypt of organoids. In (b), representative ratiometric images of mCherry-SEpHluorin ratio in a WT organoid (control) and an WT organoid treated with 5 μM EIPA from day 1 to day 3. Dashed box with red line (basal layer), cells for enlarged view. Bottom panel, enlarged views showing pHi gradient in -EIPA but not in +EIPA. Representative images are from representative 3 preparations. Col., Column. Pan., Paneth cell. In (c), values of pHi of different crypt cell types determined by calibrating fluorescent ratios (n = 3 biologically independent experiments; two-sided Mann–Whitney test). Blue, A pHi gradient; Green, attenuated pHi gradient by EIPA. Box plots are minimum to maximum, the box shows 25th-75th percentiles, and the central line is the median. p values in -EIPA: ap = 0.0001; bp = 0.0001: cp = 0.2332; dp = 0.7492; ep = 0.0002; fp = 0.0486. p values in +EIPA: gp = 9.222E-06; hp = 0.0154; ip = 0.0396; jp = 0.0468; kp = 0.2603. p values in -EIPAvs+EIPA: lp = 0.4074; mp = 0.0054; np = 0.0289; op = 0.2609; pp = 0.0032. Source data are provided as a Source Data file. In (d), representative frames (7 of 51) from 15-h time-lapse videos of crypt budding started 24 h after seeding. Arrowheads, growing crypt. BF bright-field view. Fluor. Ratio, ratiometric view of mCherry-SEpHluorin. Time-lapse video see Supplementary Movie 1. e, f pHi in the isolated crypts. In (e), representative ratometric images of freshly isolated crypts loaded with BCECF. Individual fluorescent channels (see Supplementary Fig. 1d). Ratios of BCECF were sampled from bottom, lower column, higher column, and top along the crypt axis. pHi values were calibrated from ratios (n = 2 biologically independent experiments, two-sided Welch’s t test) and plotted in (f). p values in (f), BottomvsLower Col.p = 9.753E–09; Lower Col.vsHigher Col.p = 7.204E–06; Higher Col.vsTopp = 0.0044; BottomvsTopp < 2.2E–16. All plots: *p  <  0.05, **p  <  0.01, ***p  <  0.001. n.s., not statistically significant. Source data are provided as a Source Data file. All scale bars represent 20 μm.
