Extended Data Fig. 1: Piezo1 regulates proliferation and differentiation of mobilized HSCs.
From: Shear stress governs hematopoietic stem cell fate to promote inflammation-induced aging
a, Relative Piezo1 mRNA (n = 5 mice per group; shPiezo1 #1 versus shCon P < 0.001, shPiezo1 #2 versus shCon P < 0.001) and protein (n = 3 mice per group; shPiezo1 #1 versus shCon P = 0.001, shPiezo1 #2 versus shCon P = 0.001) expression in CD34+ hCB cells. b, Current-pressure relationships for shPiezo1 #2-treated CD34+ hCB cells with Yoda1 stimulation (n = 7 cells per group; shCon+Yoda1 versus shCon+Vehicle P < 0.001). c, The average Ca2+ transient amplitude (Fmax/F0) in each CD34+ hCB cell (n = 35 cells per group; shCon+Yoda1 versus shCon+Vehicle P < 0.001). d, Schematic view of mice subjected to G-CSF. e, Cell counts of LT-HSCs in the bone marrow of Piezo1f/f;Vav mice after G-CSF treatment (n = 5 mice per group; Piezo1f/f;Vav + G-CSF versus Piezo1f/f + G-CSF P < 0.001). f, Percentages of BrdU+ and quiescent G0 LT-HSCs (n = 5 mice per group; BrdU+ LT-HSCs: Piezo1f/f;Vav + G-CSF versus Piezo1f/f + G-CSF P < 0.001; G0 LT-HSCs: Piezo1f/f;Vav + G-CSF versus Piezo1f/f + G-CSF P = 0.002). g, CFU assay of equal numbers of LT-HSCs (n = 5 mice per group; CFU-GM/G/M: Piezo1f/f;Vav + G-CSF versus Piezo1f/f + G-CSF P = 0.002). h, Schematic view of mice treated with GsMTx4 every 3 days for 2 weeks, with a total of 5 injections, in combination with G-CSF. i, Percentage of quiescent G0 LT-HSCs in blood and CFU assay of equal numbers of blood LT-HSCs (n = 5 mice per group; G0 LT-HSCs: GsMTx4+G-CSF versus Vehicle+G-CSF P = 0.044; CFU-GM/G/M: GsMTx4+G-CSF versus Vehicle+G-CSF P = 0.033). j, Schematic view of mice subjected to NE. k, Cell counts of LT-HSCs in the bone marrow of Piezo1f/f;Vav mice after NE treatment (n = 5 mice per group; Piezo1f/f;Vav + NE versus Piezo1f/f + NE P < 0.001). l, Percentages of BrdU+ and quiescent G0 LT-HSCs (n = 5 mice per group; BrdU+ LT-HSCs: Piezo1f/f;Vav + NE versus Piezo1f/f + NE P = 0.009; G0 LT-HSCs: Piezo1f/f;Vav + NE versus Piezo1f/f + NE P = 0.004). m, CFU assay of equal numbers of LT-HSCs (n = 5 mice per group; CFU-GM/G/M: Piezo1f/f;Vav + NE versus Piezo1f/f + NE P < 0.001). n, Schematic view of mice exposed to hypoxia. o, Cell counts of LT-HSCs in the bone marrow of Piezo1f/f;Vav mice exposed to hypoxia (n = 5 mice per group; Piezo1f/f;Vav+Hypoxia versus Piezo1f/f+Hypoxia P < 0.001). p, Percentages of BrdU+ and quiescent G0 LT-HSCs (n = 5 mice per group; BrdU+ LT-HSCs: Piezo1f/f;Vav+Hypoxia versus Piezo1f/f+Hypoxia P = 0.006; G0 LT-HSCs: Piezo1f/f;Vav+Hypoxia versus Piezo1f/f+Hypoxia P = 0.007). q, CFU assay of equal numbers of LT-HSCs (n = 5 mice per group; CFU-GM/G/M: Piezo1f/f;Vav+Hypoxia versus Piezo1f/f+Hypoxia P = 0.001). r, Percentage of BrdU+ LT-HSCs and CFU assay of equal numbers of LT-HSCs obtained at ZT5 and ZT13 and passed through microfluidic device (n = 5 mice per group; BrdU+ LT-HSCs: ZT5+Wc 15 μm versus ZT5+Wc 200 μm P = 0.012, ZT13+Wc 15 μm versus ZT13+Wc 200 μm P = 0.031; CFU-GM/G/M: ZT5+Wc 15 μm versus ZT5+Wc 200 μm P = 0.024, ZT13+Wc 15 μm versus ZT13+Wc 200 μm P = 0.032). s, Schematic view of the hydrostatic pressure modulation apparatus and a graph of the pressure regimes applied within the chamber. t, Percentage of BrdU+ LT-HSCs after exposure to static pressure at 40 or 60 mm Hg (n = 5 mice per group). u, Current-pressure relationships for LT-HSCs after exposure to static pressure for 6 h (n = 7 cells per group). v, Percentage of BrdU+ LT-HSCs after exposure to cyclic pressure (n = 5 mice per group; cyclic 4 h versus 0 h P = 0.010, cyclic 6 h versus 0 h P = 0.007). w, Percentage of BrdU+ LT-HSCs after exposure to cyclic pressure for 6 h (n = 5 mice per group; Piezo1f/f+Cyclic versus Piezo1f/f+Con P < 0.001). x, Current-pressure relationships for LT-HSCs after exposure to cyclic pressure for 6 h (n = 7 cells per group; Piezo1f/f+Cyclic versus Piezo1f/f+Con P < 0.001). y, Percentage of BrdU+ LK cells (n = 5 mice per group; 70 μm/70 psi versus MACS P = 0.018, 85 μm/45 psi versus MACS P = 0.015, 100 μm/20 psi versus MACS P = 0.015) and CFU assay of equal numbers of LK cells sorted by MACS or flow cytometry (n = 5 mice per group; CFU-GM/G/M: 70 μm/70 psi versus MACS P = 0.015, 85 μm/45 psi versus MACS P = 0.028, 100 μm/20 psi versus MACS P = 0.037). z, Percentage of BrdU+ LK cells (n = 5 mice per group) and CFU assay of equal numbers of LK cells from the first sorting and the second sorting by flow cytometry (n = 5 mice per group). The data are representative of three (a,b,e-g,i,k-m,o-r,t-z) and four (c) 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 (a,t,v,y), two-way repeated measures ANOVA (b,u,x), two-way ANOVA with Tukey’s post hoc test (c,e-g,k-m,o-r,w), or two-sided Student’s t test (i,z).
