Fig. 2.

Characterization of mouse intestinal organoids expressing the FRET biosensor for ERK activity. a Experimental setting for live imaging of intestinal organoids. Organoids were established from the small intestine of EKAREV-NLS mice, and cultured in media containing EGF, Noggin, and R-spondin1. Imaging was performed within 4 days of the last passage. b A representative FRET/CFP ratio image of an EKAREV-NLS organoid cultured under normal conditions. c–h Time-lapse imaging of ERK activity in the EKAREV-NLS organoids. Organoids were treated with 1 μM TPA (c–e) or 200 nM MEK inhibitor (PD0325901) (f–h) at time point 0. c, f Representative time-lapse images of organoids treated with TPA (c) or a MEK inhibitor (f) are shown. d, g Time courses of the average FRET/CFP values in the organoids (n = 90 (d) and 55 (g) cells). The FRET/CFP values in individual cells were normalized to the mean values before the treatment. e, h Bee swarm plots of the FRET/CFP values in each cell before and after the treatment (n = 90 (e) and 66 (h) cells). The FRET/CFP values in individual cells were normalized to the mean values before the treatment. i Time courses of the average ERK activity (FRET/CFP ratio) in EKAREV-NLS organoids treated with 10 μM, 1 μM, or 100 nM of a BRAF inhibitor (SB590885) (n = 30, 35, and 31 cells, respectively). Scale bars, 50 µm. Red lines represent mean. Error bars represent s.e.m. Mann–Whitney U-tests were used for comparison (e, h). *P < 0.05, **P < 0.001, ***P < 0.0001