Figure 5. Gap junction inhibitor exerted limited effects on the synchronization of Ca²⁺ dynamics.

Ca²⁺ synchronization among two or three ROIs in GCaMP6s-expressing organoids was evaluated before and after treatment with 100 µM CBX. The synchronization was unaffected between (A) SMC-SMC and (B) ICC-ICC, but was partially affected between (C) ICC–SMC. Magnified views (a, b) highlight that the preceding Ca²⁺ peak in ICC (green) observed before CBX treatment (a) was abolished after treatment (b). (D) Peak latency between ICC and SMC. Median values: Before, 1.167; CBX 20  µM, 0.701; CBX 100  µM, 0.0. Statistical significance was assessed using Welch’s t-test. ***p<0.001, n.s., p≥0.05 (p=0.27 for Before vs. CBX 20 µM). Sample sizes: Before: n = 10, peak count  = 50; CBX 20 µM: n = 5, peak count  = 34; 100 µM: n = 4, peak count  = 51, Scale bar: 50 µm (A).

Figure 5—figure supplement 1. Effects of gap junction inhibitors on organoidal contractions and ICC-SMC latency.

(A) Organoids at day 7 exhibited rhythmic contractions before and after administration of 100  µM CBX. Graphs show normalized contraction intensities visualized using the Time Measurement function. Arrowheads indicate contraction peaks defined by a peak prominence > 0.25 and a peak width ≤ 10 s. (B) Contraction intervals before and after treatment with CBX (100  µM), H₂O (vehicle control for CBX), 18β-GA (100  µM), and DMSO (vehicle control for 18β-GA). Median values: CBX, 14.0 (before), 11.9 (after); H₂O, 14.7 (before), 15.4 (after); 18β-GA, 14.7 (before), 7.7 (after); DMSO, 19.95 (before), 17.5 (after). Sample sizes and peak counts: CBX, n = 9 (before), n = 9 (after), peak count  = 150 (before), 196 (after); H₂O, n = 10 (before), n = 10 (after), peak count  = 173 (before), 171 (after); 18β-GA, n = 7 (before), n = 8 (after), peak count  = 129; DMSO, n = 7 (before), n = 6 (after), peak count  = 193 (before), 92 (after). (C) Contraction intervals at day 7 in organoids cultured with CBX, H₂O, 18β-GA, and DMSO from day 3 to day 7. Median values: H₂O, 22.75; CBX, 32.5; DMSO, 18.85; 18β-GA, 27.95. Sample sizes and peak counts: H₂O, n = 9, peak count  = 136; CBX, n = 11, peak count  = 135; DMSO, n = 9, peak count  = 209; 18β-GA, n = 18, peak count  = 268. (D) Contractions of cultured embryonic heart cells. Graphs show normalized contraction intensities using the Time Measurement function. The box plot shows the number of contractions per 5 min. Median values: CBX, 30.75 (before), 15 (CBX 100 µM), 55.75 (washout); 18β-GA: 45.5 (before), 8.5 (18β-GA 100 µM), 51 (washout). Statistical significance was assessed using Welch’s t-test. **p<0.01, *p<0.05; n.s., p≥0.05 (p=0.13 for Before vs. washout of CBX; p=0.26 for Before vs. washout of 18β-GA). Sample sizes: CBX, n = 4; 18β-GA, n = 4. (E) Peak latency of Ca²⁺ transients between ICC and SMC before and after administration of 18β-GA (20  µM, 100  µM). Statistical significance was assessed using Welch’s t-test. n.s., p=0.27 for Before vs. 18β-GA 20 µM; p=0.25 for Before vs. 18β-GA 100 µM; p=0.46 for 18β-GA 20 µM vs. 100 µM. Sample sizes and peak counts: Before, n = 5, peak count  = 62; 18β-GA 20  µM, n = 3, peak count  = 52; 18β-GA 100  µM, n = 2, peak count  = 57. Scale bar: 50  µm (A).

Figure 5—video 1. Ca²⁺ dynamics in a gut contractile organoid with CBX.

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Time-lapse images were obtained with 700 ms intervals for 5 min. This video corresponds to Figure 5. Scale bars: 50 µm.

Figure 5—video 2. Periodic contractions of a day 7 organoid with CBX.

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Time-lapse images were obtained with 700 ms intervals for 5 min. This video corresponds to Figure 5—figure supplement 1. Scale bars: 50 µm.