Journal: International Journal of Medical Sciences

Article Title: HK2-mediated Glycolysis Inhibits Mineralization of Cementoblasts Under Compression by Suppressing the Piezo1/Wnt Signaling

doi: 10.7150/ijms.109287

Figure Lengend Snippet: Compression inhibited cementoblasts' mineralization and Piezo1 expression. (A) The relative mRNA levels of Piezo1 were detected by qRT-PCR after cementoblasts were subjected to 2.0 g/cm² compression for 12 hours under distinct buffer membranes. (B) and (C) The relative mRNA and protein levels of Piezo1, RANKL, OPG, RANKL/OPG, Runx2, OSX, and OPN were identified by qRT-PCR and WB. The blot images represented data from 3 independent experiments. The data are presented as mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Article Snippet: The PVDF membranes were blocked at room temperature with 5% bovine serum albumin (BSA) for 1 hour, followed by incubation overnight at 4°C with primary antibodies against Piezo1 (1:1000, ABclonal, China), GLUT1 (1:5000, ABclonal, China), HK2 (1:1000, Abcam, UK), PFKFB3 (1:2000, Abcam, UK), LDHA (1:2000, Abcam, UK), β-catenin (1:1000, ABclonal, China), TCF1 (1:1000, ABclonal, China), LEF1 (1:1000, ABclonal, China), RANKL (1:1000, Proteintech, USA), OPG (1:1000, Proteintech, USA), Runx2 (1:1000, Cell Signaling Technology, USA), OSX (1:1000, Cell Signaling Technology, USA), and OPN (1:1500, Cell Signaling Technology, USA), all from rabbit sources.

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Medical Sciences

Article Title: HK2-mediated Glycolysis Inhibits Mineralization of Cementoblasts Under Compression by Suppressing the Piezo1/Wnt Signaling

doi: 10.7150/ijms.109287

Figure Lengend Snippet: Piezo1 mediated mechanotransduction in cementoblasts and promoted mineralization. (A) The knockdown efficiency of the three Piezo1 siRNAs was confirmed using qRT-PCR and WB. (B) The impact of varying doses of Yoda1 on cell viability after 24h was evaluated by the CCK-8 assay. (C) Intracellular calcium concentration was detected by the Fluo-4 AM calcium ion fluorescence probe. Images were captured by fluorescence microscopy. Scale bar = 50μm. (D) and (E) The relative mRNA and protein levels of RANKL, OPG, RANKL/OPG, Runx2, OSX, and OPN were identified using qRT-PCR and WB. The blot images represented data from three independent experiments. (F) Von Kossa (VK), Alizarin Red S (ARS) (after 3 weeks of mineralization induction) and alkaline phosphatase staining (after 2 weeks of mineralization induction). Scale bar = 10μm. The data are presented as mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Con: Control, Pz1: Piezo1, F: Force.

Article Snippet: The PVDF membranes were blocked at room temperature with 5% bovine serum albumin (BSA) for 1 hour, followed by incubation overnight at 4°C with primary antibodies against Piezo1 (1:1000, ABclonal, China), GLUT1 (1:5000, ABclonal, China), HK2 (1:1000, Abcam, UK), PFKFB3 (1:2000, Abcam, UK), LDHA (1:2000, Abcam, UK), β-catenin (1:1000, ABclonal, China), TCF1 (1:1000, ABclonal, China), LEF1 (1:1000, ABclonal, China), RANKL (1:1000, Proteintech, USA), OPG (1:1000, Proteintech, USA), Runx2 (1:1000, Cell Signaling Technology, USA), OSX (1:1000, Cell Signaling Technology, USA), and OPN (1:1500, Cell Signaling Technology, USA), all from rabbit sources.

Techniques: Knockdown, Quantitative RT-PCR, CCK-8 Assay, Concentration Assay, Fluorescence, Microscopy, Staining, Control

Journal: International Journal of Medical Sciences

Article Title: HK2-mediated Glycolysis Inhibits Mineralization of Cementoblasts Under Compression by Suppressing the Piezo1/Wnt Signaling

doi: 10.7150/ijms.109287

Figure Lengend Snippet: The Wnt/β-catenin signaling promoted mineralization of cementoblasts. (A) The effects of different concentrations of DKK1 and LiCl on cell viability after 24 hours was evaluated using the CCK-8 assay. (B) and (C) The relative mRNA and protein levels of β-catenin and its target transcription factors TCF1 and LEF1 were detected by qRT-PCR and WB. (D) and (E) The relative mRNA and protein levels of RANKL, OPG, RANKL/OPG, Runx2, OSX, and OPN were identified by qRT-PCR and WB. The blot images represented data from 3 independent experiments. The data are presented as mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Con: Control, F: Force.

Article Snippet: The PVDF membranes were blocked at room temperature with 5% bovine serum albumin (BSA) for 1 hour, followed by incubation overnight at 4°C with primary antibodies against Piezo1 (1:1000, ABclonal, China), GLUT1 (1:5000, ABclonal, China), HK2 (1:1000, Abcam, UK), PFKFB3 (1:2000, Abcam, UK), LDHA (1:2000, Abcam, UK), β-catenin (1:1000, ABclonal, China), TCF1 (1:1000, ABclonal, China), LEF1 (1:1000, ABclonal, China), RANKL (1:1000, Proteintech, USA), OPG (1:1000, Proteintech, USA), Runx2 (1:1000, Cell Signaling Technology, USA), OSX (1:1000, Cell Signaling Technology, USA), and OPN (1:1500, Cell Signaling Technology, USA), all from rabbit sources.

Techniques: CCK-8 Assay, Quantitative RT-PCR, Control

Journal: International Journal of Medical Sciences

Article Title: HK2-mediated Glycolysis Inhibits Mineralization of Cementoblasts Under Compression by Suppressing the Piezo1/Wnt Signaling

doi: 10.7150/ijms.109287

Figure Lengend Snippet: HK2-mediated glycolysis inhibited cementoblasts' mineralization by suppressing Piezo1 expression. (A) The knockdown efficiency of the three HK2 siRNAs was verified by qRT-PCR and WB. (B) Glucose consumption and lactate production in cementoblasts, normalized to cell number. (C) and (D) The relative mRNA and protein levels of Piezo1, RANKL, OPG, RANKL/OPG, Runx2, OSX, and OPN were identified by qRT-PCR and WB. The blot images represented data from 3 independent experiments. (E) Immunofluorescence of Piezo1 with DAPI counterstaining. Scale bar = 20μm. (F) Von Kossa (VK), Alizarin Red S (ARS) (after 3 weeks of mineralization induction) and alkaline phosphatase staining (after 2 weeks of mineralization induction). Scale bar = 10μm. The data are presented as mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Con: Control, F: Force.

Article Snippet: The PVDF membranes were blocked at room temperature with 5% bovine serum albumin (BSA) for 1 hour, followed by incubation overnight at 4°C with primary antibodies against Piezo1 (1:1000, ABclonal, China), GLUT1 (1:5000, ABclonal, China), HK2 (1:1000, Abcam, UK), PFKFB3 (1:2000, Abcam, UK), LDHA (1:2000, Abcam, UK), β-catenin (1:1000, ABclonal, China), TCF1 (1:1000, ABclonal, China), LEF1 (1:1000, ABclonal, China), RANKL (1:1000, Proteintech, USA), OPG (1:1000, Proteintech, USA), Runx2 (1:1000, Cell Signaling Technology, USA), OSX (1:1000, Cell Signaling Technology, USA), and OPN (1:1500, Cell Signaling Technology, USA), all from rabbit sources.

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Immunofluorescence, Staining, Control