Fig 1: The expression and function of Piezo1 in the pre-osteoblast cell line MC3T3-E1.(a) Representative traces of poking-induced inward currents recorded at -60 mV in the indicated cell lines. (b) Scatter plots of the maximal poking-induced currents in the indicated cell lines. (c) QRT-PCR analysis of Piezo1 mRNA level in the indicated cell lines. (d) Representative traces of poking-induced inward currents recorded at -60 mV in MC3T3-E1 under the indicated conditions. 'siRNA' indicates the siRNA-mediated knockdown of Piezo1. NC, Piezo1 negative-control siRNA. GsMTX4 is a relatively specific blocker of the Piezo channel family. (e) Scatter plots of the maximal poking-induced currents in MC3T3-E1 cells under the indicated conditions. (f, g) QRT-PCR analysis of Piezo1 mRNA level (f) and western blot analysis of Piezo1 protein level (g) in MC3T3-E1 cells transfected with control or Piezo1 siRNA for 48 hr. (h) QRT-PCR analysis of Alp, Bglap, and Col1a1 mRNA levels in MC3T3-E1 cells transfected with control or Piezo1 siRNA for 48 hr. (i) Representative images of Alp staining in MC3T3-E1 cells transfected with control or Piezo1 siRNA for 48 hr. Scale bar, 5 mm. *, p<0.05; **, p<0.01; ***, p<0.001.10.7554/eLife.47454.005Figure 1—source data 1.The expression and function of Piezo1 in pre-osteoblast cell line MC3T3-E1.
Fig 2: Expression and function of Piezo1 in primary osteoblasts isolated from Piezo1fl/fl and Piezo1Ocn/Ocn mice.(a) QRT-PCR analysis of Piezo1 mRNA level in bone and other tissues from WT mice. (b) QRT-PCR analysis of Piezo1 mRNA level in different tissues from Piezo1fl/fl or Piezo1Ocn/Ocn mice. The Piezo1 mRNA level in all tissues from Piezo1Ocn/Ocn mice was normalized to that in Piezo1fl/fl mice. (c) Western blot analysis of Piezo1 protein level in bone tissues from Piezo1fl/fl and Piezo1Ocn/Ocn mice. (d) QRT-PCR analysis of Piezo1 mRNA level in primary osteoblasts isolated from Piezo1fl/fl or Piezo1Ocn/Ocn mice and cultured with osteogenic medium for 5 days. (e) Western blot analysis of Piezo1 protein level in primary osteoblasts cultured with osteogenic medium for 5 days. (f) Representative traces of poking-induced inward currents recorded at -60 mV in primary osteoblasts isolated from Piezo1fl/fl or Piezo1Ocn/Ocn mice. (g, h) Scatter plots of the maximal poking-induced currents (g) and inactivation tau (h) in primary osteoblasts isolated from Piezo1fl/fl or Piezo1Ocn/Ocn mice. (i) Average single-cell Ca2+ imaging traces of Piezo1fl/fl or Piezo1Ocn/Ocn osteoblasts showing the 340/380 ratio of the Ca2+-sensitive Fura-2 dye in response to the application of 30 µM Yoda1. (j) Scatter plot of baseline and Yoda1-induced Fura-2 amplitude changes in Piezo1fl/fl or Piezo1Ocn/Ocn osteoblasts. (k) Western blot analysis of p-CaMKII, CaMKII, p-Creb, Creb, Runx2 and Atf4 proteins in primary osteoblasts cultured with osteogenic medium for 5 days. (l) QRT-PCR analysis of Alp, Bglap and Col1a1 mRNA levels in primary osteoblasts cultured with osteogenic medium for 5 days. (m) Representative images of Alp staining in primary osteoblasts cultured with osteogenic medium for 5 days. Scale bar, 5 mm. The staining data were confirmed by three repeated tests. All data are the mean ± s.e.m. from three independent experiments. **, p<0.01; ***, p<0.001.10.7554/eLife.47454.009Figure 2—source data 1.The expression and function of Piezo1 in primary osteoblasts isolated from Piezo1fl/fl and Piezo1Ocn/Ocn mice.
Fig 3: Oroxylin attenuates bone loss in OVX mice.(A) Representative micro-CT images of femora of sham, OVX, and OVX + oroxylin A groups. Scale bars, 1 mm. (B) Quantitative micro-CT of BMD, BV/TV, and Tb.N of femora. n = 6 per group. (C) Quantitative micro-CT of the cortical thickness of femora. n = 6 per group. (D and E) Hematoxylin and eosin staining of femora with quantification of Tb area/tissue area × 100%. Scale bar, 500 µm. n = 5 per group. (F) Angiographic images of femora. Scale bar, 20 µm. n = 5 per group. (G) Quantitative analyses of vessel volume and vessel surface. n = 5 per group. (H) Representative images of immunostaining of EMCN (red), CD31 (green), and EmcnhiCD31hi (yellow) cells on trabecular bone and periosteal bone. Scale bar, 50 µm. n = 5 per group. (I) The percentage of EmcnhiCD31hi area on trabecular bone and periosteal bone. n = 5 per group. (J) Flow cytometry plots with the percentage of CD31hiEmcnhi endothelial cells in total bone marrow cells. n = 3 per group. (K to M) Quantification of mineral apposition rate, bone formation rate per bone surface, mineralizing surface per bone surface in calcein double labeling. n = 5 per group. (N) Serum PDGF-BB and VEGF levels. n = 6 per group. (O) Serum CTX-1 and TRAcp5b levels. n = 6 per group. (P) Serum OCN levels. n = 6 per group. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 4: Osteoporosis bone specimens have decreased Piezo1 expression and correlated defective osteoblast function.(a) QRT-PCR analysis of Piezo1 mRNA level in bone specimens from two T-score groups. T > -2.5 group, n = 10, and T = -2.5 group, n = 10. (b) Western blot analysis of Piezo1 protein level in bone specimens from two T-score groups. T > -2.5 group, n = 7, and T=- 2.5 group, n = 7. Quantification of Piezo1 protein level was normalized to GAPDH. (c) Correlation analysis between Piezo1 level and the levels of ALP, BGLAP or COL1a1. T > -2.5 group, n = 10, and T = -2.5 group, n = 10. (d) Correlation analysis between Piezo1 levels and the levels of CTSK (cathepsin K), ACP5 (acid phosphatase 5) or MMP9 (matrix metallopeptidase 9). T > -2.5 group, n = 10, and T = -2.5 group, n = 10. (e) Correlation analysis between Piezo1 levels and the level of DMP1 (dentin matrix acidic phosphoprotein 1 ) or SOST (sclerostin). All data are the mean ± s.e.m. *, p<0.05; **, p<0.01; ***, p<0.001.10.7554/eLife.47454.021Figure 7—source data 1.The data and statistical analysis of the relationship between Piezo1 expression levels and bone formation in human specimens.
Fig 5: Bone biomarker and short-chain fatty acid profiling in serum of the ISS flight cohort versus the ISS_G ground control cohort(A) ELISA analysis of bone biomarkers OCN, P1NP, and TRACP in the ISS cohort versus the ISS_G. Unpaired Student’s t test statistical analysis was performed for comparisons of the ISS cohort versus the ISS_G. Statistical significance is indicated accordingly.(B and C) Absolute quantification of R/S-2,3-butanediol and meso-2,3-butanediol and short-chain fatty acids detected via targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). Unpaired Student’s t test statistical analysis was performed for comparisons between ISS and ISS_G cohorts. Abundance and individual statistical indications are listed in Tables S12–S14 and statistical significance is indicated accordingly.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for Mouse Osteocalcin ELISA Kit (Colorimetric)