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. Author manuscript; available in PMC: 2022 Sep 2.
Published in final edited form as: Cell Stem Cell. 2021 Apr 28;28(9):1516–1532.e14. doi: 10.1016/j.stem.2021.04.002

Figure 7. Piezo channel activity is necessary for inflation-mediated fission.

Figure 7.

(A) Experimental design for imaging intracellular calcium dynamics after stimulating or inhibiting luminal inflation or Piezo channel activity.

(B) Top: the calcium dye CA-630 shows increased intensity in the epithelium surrounding an inflated (inf.) bud compared with a collapsed (coll.) bud. Bottom: after forskolin treatment, the previously coll. bud (marked by an asterisk) shows increased CA-630 intensity.

(C) Representative images of calcium imaging under perturbation.

(D) Quantification of mean CA-630 dye intensity under different conditions. F, C, G/F as defined in (C) (n = 29 no-drug controls, n = 64 no-dye controls, n = 29 forskolin-treated organoids, n = 31 CFTRinh-172-treated organoids, n = 33 GdCl3 + forskolin-treated organoids; 2 biological replicates per condition). Error bars indicate SEM. One-sided t test: *p < 0.05, **p < 0.01, ***p < 0.001.

(E) Experimental designs to quantify SCZ fission after sustained inhibition or acute activation of Piezo ion channels.

(F) Channel inhibition significantly reduces the number of fission events per organoid compared with vehicle control (n = 70 organoids in DMSO across 6 replicates, n = 34 in GdCl3 across 3 replicates, n = 24 in GsMTx4 across 3 replicates).

(G) Treatment with the Piezo agonist Yoda1 does not significantly alter fission frequencies (n = 61 in DMSO across 6 replicates, n = 38 in Yoda1 across 6 replicates).

(H) Piezo channel inhibition weakly suppresses inflation. Data are from same organoids as in (F).

(I) Experimental design to test for epistasis between inflation and Piezo channel activity on SCZ fission rates.

(J) and K) Dual treatment with the Piezo channel inhibitor GdCl3 and the CFTR agonist forskolin (G+F) increases organoid inflation, but the SCZ fission rate is reduced, consistent with Piezo channel activity acting downstream of inflation to mediate SCZ fission. Experiments were carried out separately from those in (E)–(H), using smaller organoid fragments that exhibited lower inflation and fission rates (n = 59 organoids in DMSO, n = 53 in GdCl3 + forskolin, 6 replicates each).