Extended Data Fig. 3: Piezo1 regulates human hematopoietic progenitor cell proliferation and differentiation.

a, Schematic view of Piezo1 knockdown or Yoda1 stimulation in CD34⁺ cells from mobilized human peripheral blood. b, Relative Piezo1 mRNA (n = 5 donors per group; shPiezo1 #1 versus shCon P < 0.001, shPiezo1 #2 versus shCon P < 0.001) and protein (n = 3 donors per group; shPiezo1 #1 versus shCon P < 0.001, shPiezo1 #2 versus shCon P < 0.001) expression in human peripheral blood CD34⁺ (CD34⁺ hPB) cells. c,d, Protein expression of Piezo1 in shPiezo1 #1-treated CD34⁺ hPB cells with Yoda1 stimulation (n = 3 donors per group; shPiezo1 #1+Vehicle versus shCon+Vehicle P = 0.021). e, Percentages of BrdU⁺ CD34⁺ hPB cells (n = 5 donors per group; shCon+Yoda1 versus shCon+Vehicle P = 0.013). f, SiC division assay of CD34⁺ hPB cells (n = 5 donors per group; divided cells: shCon+Yoda1 versus shCon+Vehicle P = 0.005). g, Protein expression of Piezo1 in shPiezo1 #2-treated CD34⁺ hPB cells with Yoda1 stimulation (n = 3 donors per group; shPiezo1 #1+Vehicle versus shCon+Vehicle P = 0.004). h, Percentages of BrdU⁺ CD34⁺ hPB cells (n = 5 donors per group; shCon+Yoda1 versus shCon+Vehicle P = 0.015). i, SiC division assay of CD34⁺ hPB cells (n = 5 donors per group; divided cells: shCon+Yoda1 versus shCon+Vehicle P = 0.001). j, Schematic view of Piezo1 knockdown or Yoda1 stimulation in CD34⁺ hCB cells. k, Protein expression of Piezo1 in shPiezo1 #1-treated CD34⁺ hCB cells with Yoda1 stimulation (n = 3 donors per group; shPiezo1 #1+Vehicle versus shCon+Vehicle P = 0.030). l, Percentages of BrdU⁺ CD34⁺ hCB cells (n = 5 donors per group; shCon+Yoda1 versus shCon+Vehicle P = 0.018). m, SiC division assay of CD34⁺ hCB cells (n = 5 donors per group; divided cells: shCon+Yoda1 versus shCon+Vehicle P = 0.008). n, Protein expression of Piezo1 in shPiezo1 #2-treated CD34⁺ hCB cells with Yoda1 stimulation (n = 3 donors per group; shPiezo1 #2+Vehicle versus shCon+Vehicle P = 0.004). o, Percentages of BrdU⁺ CD34⁺ hCB cells (n = 5 donors per group; shCon+Yoda1 versus shCon+Vehicle P = 0.020). p, SiC division assay of CD34⁺ hCB cells (n = 5 donors per group; divided cells: shCon+Yoda1 versus shCon+Vehicle P = 0.014). q, Serial colony-forming unit assays of shPiezo1 #1-treated CD34⁺ hCB cells with Yoda1 stimulation (n = 5 donors per group; #1° colony numbers: shCon+Yoda1 versus shCon+Vehicle P = 0.016; #2° colony numbers: shCon+Yoda1 versus shCon+Vehicle P = 0.027). r, CFU assay of equal numbers of CD34⁺ hCB cells (n = 5 donors per group; CFU-GM/G/M: shCon+Yoda1 versus shCon+Vehicle P = 0.013). s,t, Serial colony-forming unit assays of shPiezo1 #2-treated CD34⁺ hCB cells with Yoda1 stimulation (n = 5 donors per group; #1° colony numbers: shCon+Yoda1 versus shCon+Vehicle P = 0.032; #2° colony numbers: shCon+Yoda1 versus shCon+Vehicle P = 0.026). u, CFU assay of equal numbers of CD34⁺ hCB cells (n = 5 donors per group; CFU-GM/G/M: shCon+Yoda1 versus shCon+Vehicle P = 0.008). v, Percentage of BrdU⁺ CD34⁺ hCB cells (n = 5 donors per group; 70 μm/70 psi versus MACS P = 0.012, 85 μm/45 psi versus MACS P = 0.014, 100 μm/20 psi versus MACS P = 0.016) and CFU assay of equal numbers of CD34⁺ hCB cells sorted by MACS or flow cytometry (n = 5 donors per group; CFU-GM/G/M: 70 μm/70 psi versus MACS P = 0.012, 85 μm/45 psi versus MACS P = 0.019, 100 μm/20 psi versus MACS P = 0.019). w, Percentage of BrdU⁺ CD34⁺ hCB cells (n = 5 donors per group) and CFU assay of equal numbers of CD34⁺ hCB cells from the first sorting and the second sorting by flow cytometry (n = 5 donors per group). The data are representative of two (f,i,m,p) and three (b-e,g,h,k,l,n,o,q-w) independent experiments. Data are the mean value ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (b,v), two-way ANOVA with Tukey’s post hoc test (d-i, k-u), or two-sided Student’s t test (w).

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