Figure 8. Piezo1 was required to sense ECM stiffness and upregulate Yap1 and Ctnnb1 activities in primary mouse BMSCs.

(a) Yap1 immunostaining of Piezo1^f/f;Piezo2^f/f BMSCs seeded on matrices with the indicated stiffness after Ad-GFP or Ad-Cre infection. Scale bars: 100 μm. (b) BMSC were infected with the indicated Ad-virus, plated on stiff (40 kPa) or soft (1 kPa) substrates and induced to differentiate into osteoblasts for 6 days. Representative alkaline phosphatase stainings images were shown. Scale bar: 100 mm. As shown previously (Dupont et al., 2011). Osteogenic differentiation was quantified by the alkaline-phosphatase-positive area determined with ImageJ as the number of blue pixels across the picture. This value was normalized to the number of cells (Hoechst/nuclei) for each picture (arbitrary units). RT-PCR anlaysis of BMSCs grown on the indicated hydrogels was shown below. (c) Alkaline phosphatase staining and qPCR analyses of primary mouse BMSCs treated with shaking with or without a Ppp3ca inhibitor CsA (n = 3, means ± SD). (d) Alkaline phosphatase staining and qPCR analyses of BMSCs treated with Yoda1 and CsA (n = 3, means ± SD). *p<0.05, **p<0.01, ***p<0.001, one-way ANOVA followed by Tukey’s multiple comparisons tests (b–d) (Figure 8—source data 1).

Figure 8—figure supplement 1. Piezo mutant primary BMSCs were defective in mechanotransduction.

(a) Representative images of the GCaMP5G reporter and the internal tdTomato fluorescent control in BMSCs of the indicated genotypes after Ad-Cre infection. The BMSCs were cultured on matrices with the indicated stiffness. While there was no difference in the tdTomato fluorescent intensity, Ca2⁺ signaling activity indicated by GFP intensity was increased in cells on stiffer matrix and Piezo1-deficient BMSCs were less spreading with weaker GFP signals on both soft and stiff matrix. Scale bar: 100 μm. (b) Ptk2 (Fak) phosphorylation in the Ad-Cre-induced Piezo1/2 deficient BMSCs was reduced compared to the Ad-GFP infected BMSCs, suggesting focal adhesion was reduced in the absence of Piezo1/2. (c) Fluorescent immunostaining of pPtk2 in mouse primary BMSCs. Activation of Piezo1 by Yoda1 treatment increased pPtk2 staining (Figure 8—figure supplement 1—source data 1).