Extended Data Figure 6. Piezo over-expression increases cytosolic Ca²⁺ level which further triggers proliferation of ISCs but not EBs.

a. Overexpression of Piezo^GFP in esg+ cells (esg>Gal4/UAS-Piezo^GFP; UAS-RGECO) at 32°C causes an increase in cytosolic Ca²⁺ (indicated by the calcium reporter RGECO) compared to control (esg>Gal4/UAS-GFP; UAS-RGECO). Representative images from three short time-lapse imaging of cultured fly midguts were shown. Scale bar: 50 μm. b. Typical traces of Ca²⁺ oscillations in esg⁺ cells of midgut from either control or Piezo^GFP flies from three independent replicates. c. Ca²⁺ oscillation frequency of esg⁺ cells from either control or Piezo^GFP midguts. Data of 27 cells from three replicates for each condition were shown. d. Statistics for average RGECO signal intensity in all GFP⁺ cells (blue) and percentage of Ca²⁺ positive cells (signal higher than 3X s.d. of background) compared to total GFP⁺ cells (orange). Signal intensities were calculated from 10,000 μm² regions: n=17 (control), n=22 (Piezo^GFP) from three independent experiments. e, Bleomycin (Bleo, 10ug/ml) (5 days treatment) triggers a significant increase of esg⁺ cell and EE cells in both WT and Piezo^KO flies. Represented images from three independent replicates were shown. f, Images of live midguts from WT and Piezo^KO flies. Both flies were fed on food containing Bleomycin for 3 days before imaging. g,h. Traces of Ca²⁺ oscillations in Dl⁺ stem cells from WT and Piezo mutant flies fed on Bleomycin for 4–5 days. Bleomycin treatment causes some stem cells to maintain constant high Ca²⁺ levels, while others show reduced oscillation frequency but increased average GCaMP/RFP intensity ratio (G/R ratio). These data show that tissue damage by Bleomycin triggers stem cell proliferation, EE production, and an increase of cytosolic Ca²⁺, independent of Piezo. 30 cells from n=4 (control), n=4 (Bleo⁺), and n=5 (Piezo^KO + Bleo⁺) independent guts were plotted. i. Overexpression of Piezo^GFP in esg⁺ cells (32°C) increases the ratio of Dl⁺ cells (labeled by Dl-lacZ) within the esg⁺ population. j. Piezo overexpression promotes Dl⁺ stem cells ratio in esg⁺ cells. Ratio between Dl⁺ and esg⁺ cells within 10,000 μm² regions: n=21 (control) and n=22 (Piezo^GFP) from two independent replicates, are analyzed. k,l. Overexpressing Piezo or knocking down Serca in Su(H)Gbe⁺ EB cells showed no significant phenotype, suggesting that their effect may be blocked by high Notch activity. Number of midgut areas quantified: n=18 (control), n=20 (Serca-i), n=16 (Piezo^GFP). Data are expressed as mean + s.e.m. values. P-values are calculated from two-tailed Student t-test with unequal variance. Scale bar: a,e,f, 50 μm; i, 20 μm; k, 50 μm.