Extended Data Figure 5: Prolonged increase of stem-cell proliferation may reduce EE cell number.
From: Mechanical regulation of stem-cell differentiation by the stretch-activated Piezo channel
a, Fly midguts of each indicated genotype/condition were analysed after incubation for 5 and 10 days at 32 °C. Esg+ cells (GFP+, green) and EE cells (Pros+, red). Representative images from two independent replicates. b, Quantification of mitosis (pH3+ cell number) of midguts from flies expressing GFP only (control, n = 16 (5 days), n = 16 (10 days)), full-length Stim (StimOE, n = 15 (5 days), n = 17 (10 days), SERCA-i (n = 18 (5 days), n = 16 (10 days)), PiezoOE (n = 17 (5 days), n = 18 (10 days)), PMCA-i (n = 15 (5 days), n = 15 (10 days)), and flies fed bleomycin-containing food (regular food + 10 μg ml−1 bleomycin, n = 15 (5 days), n = 13 (10 days)). c, Quantification of Pros+ EE cell number from 10,000-μm2 regions: n = 31 (5 days), n = 30 (10 days) (control); n = 30 (5 days), n = 32 (10 days) (StimOE); n = 30 (5 days), n = 30 (10 days) (SERCA-i); n = 31 (5 days), n = 32 (10 days) (PiezoOE); n = 32 (5 days), n = 31 (10 days) (PMCA-i); n = 29 (5 days), n = 28 (10 days) (Bleo+). Bleomycin treatment or PMCA-i significantly reduced the number of EEs. This reduction is primarily due to increased turnover of EEs, as blocking cell mitosis for 5 days had no significant effect on EE cell number (Extended Data Fig. 7). The differences between stem-cell proliferation and EE differentiation may be due to a different level of cytosolic Ca2+ increase and the Ca2+ depletion in the ER store. d, Change of Piezo+ cells and EEs after 5 and 10 days of control (5% sucrose) or bleomycin (5% sucrose plus 10 μg ml−1 bleomycin) treatment. Representative images from two independent replicates. e, Quantification of Piezo+ cells and EEs from 10–15 midguts for each condition. Both Piezo+ cells and EEs number increased after 5 days of bleomycin treatment, and significantly decreased after 10 days of treatment. Cell numbers were quantified within a 10,000-μm2 area, except for pH3, which is quantified from the whole midgut. All data are mean + s.e.m. P values are from a two-tailed Student’s t-test with unequal variance. Scale bars, 50 μm.
