Journal: Cell Death & Disease

Article Title: P53 and Parkin co-regulate mitophagy in bone marrow mesenchymal stem cells to promote the repair of early steroid-induced osteonecrosis of the femoral head

doi: 10.1038/s41419-020-2238-1

Figure Lengend Snippet: a , b 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) detection of reactive oxygen species (ROS; n = 4). c , d TUNEL–DAPI detection of deoxyribonucleic acid (DNA) damage and apoptosis ( n = 4). e , f Annexin V–FITC and propidium iodide (PI) detection of apoptosis ( n = 4). g , h Detection of β-gal activity by β-gal staining ( n = 4). i – k Western blot analysis of P16 and P21 expression ( n = 3). In ( b ), ( d ), ( f ), ( h ), and ( j – k ), data are presented as means ± SD. Statistical significances were calculated by Student’s t test. Data were compared with the control group: * P < 0.05. H 2 O 2 was used to simulate OS; L-DMEM was used as control. TUNEL = terminal deoxynucleotidyl transferase deoxyuridine-5′-triphosphate nick end labeling; DAPI = 4′,6-diamidino-2-phenylindole; FITC = fluorescein isothiocyanate; P16 = protein 16; P21 = protein 21; β-gal = β-galactosidase.

Article Snippet: We used rabbit anti-P53 (1:1000; ab154036; Abcam, Cambridge, UK), rabbit anti-Parkin (1:300; BA1682-1; Boster Biological Technology, Ltd., Wuhan, China), rabbit anti-VDAC1 (1:1000; ab34726; Abcam), mouse anti-ubiquitin (1:1000; ab52664; Abcam), rabbit anti-NDUFA9 (1:1000; ab128744; Abcam), mouse anti-mitochondrial 70 kDa heat shock protein (mtHsp70; 1:2000; H5147; Sigma-Aldrich), rabbit anti-P21 (1:1000; bs-10129R; Bioss, Shanghai, China), rabbit anti-P16 (1:1000; A00016 -3; Boster), rabbit anti-OPG (1:1500; bs-20624R; Bioss), mouse anti-OCN (1:1000; ab13420; Abcam), rabbit anti-Runx2 (1:1000; AV36678; Sigma-Aldrich), and rabbit anti-β-actin (1:3000; ab8227; Abcam) for primary-antibody response; and horseradish peroxide (HRP)-conjugated goat anti-rabbit and goat anti-mouse immunoglobulin G (IgG; Beyotime) for secondary-antibody response.

Techniques: TUNEL Assay, Activity Assay, Staining, Western Blot, Expressing, End Labeling

Journal: BMC Molecular and Cell Biology

Article Title: Compression and tension variably alter Osteoprotegerin expression via miR-3198 in periodontal ligament cells

doi: 10.1186/s12860-019-0187-2

Figure Lengend Snippet: miR-3198 gain-of-function and loss-of-function experiments. Results of real-time RT-PCR analysis for miR-3198 ( a , b ), OPG and RANKL ( c , d ) expression in HPL cells after transfection of miR-3198 inhibitor ( a , c ) and miR-3198 mimic ( b , d ). n = 3. Biological triplicated. Fold change from the control is shown. Open bar indicates the fold change of OPG expression, and close bar indicates that of RANKL expression ( c , d ). The concentrations of OPG as measured by ELISA after transfection of miR-3198 inhibitor ( e ) and miR-3198 mimic ( f ) are shown ( n = 3). Results of real-time PCR analysis for miR-1207 ( g , h ), OPG and RANKL ( i , j ) expression in HPL cells under the transfection of miR-1207 inhibitor ( g , i ) and miR-1207 mimic ( h , j ). n = 3. Biological triplicated. Fold changes from the control are shown. * indicates P < 0.05 versus control, and NS indicating there was no significant difference between samples

Article Snippet: The concentration of OPG in the culture supernatant was measured using an OPG ELISA kit (Boster Biological Technology, Pleasanton, CA), according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: BMC Molecular and Cell Biology

Article Title: Compression and tension variably alter Osteoprotegerin expression via miR-3198 in periodontal ligament cells

doi: 10.1186/s12860-019-0187-2

Figure Lengend Snippet: miR-3198 regulates the mechanical stress-mediated change of OPG expression. Results of real-time RT-PCR analysis for OPG and RANKL expression in HPL cells in the compression ( a ) and tension ( b ) experiments. n = 3. Biological triplicated. Fold change from the control are shown. Cont, control; Inh, transfection of miR-3198 inhibitor; Mimic, transfection of miR-3198 mimic; press, compression; tens, tension; TF, transfection. Also shown are the OPG concentrations measured by ELISA in the compression ( c ) and tension ( d ) experiments ( n = 3). * indicates P < 0.05 versus control. † indicates P < 0.05 between samples. NS indicates there was no significant difference between samples. e and f Representative image of the western blotting for OPG was shown. g and h Relative band intensity of the western blotting for OPG. *: P < 0.05 versus control. †: P < 0.05 between the groups. NS, no significant difference between the samples

Article Snippet: The concentration of OPG in the culture supernatant was measured using an OPG ELISA kit (Boster Biological Technology, Pleasanton, CA), according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Control, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot

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

Journal: Cell death discovery

Article Title: Endogenous osteoprotegerin (OPG) represses ERα and promotes stemness and chemoresistance in breast cancer cells.

doi: 10.1038/s41420-024-02151-8

Figure Lengend Snippet: Fig. 7 OPG downregulation inhibits the breast carcinogenesis of breast cancer cells. MDA-MB-231 and BT-20 cells were transfected with OPG siRNA (OPG-si), while a scrambled sequence was used as control (Ctrl). A Western blot images with GAPDH used as internal control. The numbers underneath each band represent fold changes relative to the control (Ctrl) after normalization to GAPDH. Phosphorylated protein fractions were determined by quantification and normalization against their relative non-phosphorylated forms (B) Cellular analysis using the real-time xCELLigence RTCA System. C Cells (2 × 105) were harvested and stained with propidium iodide (PI), and were analyzed by flow cytometry. The fractions of cells in each phase of the cell cycle are depicted in the boxes. D Cells (1 × 103) were seeded in ultra-low attachment 96‐well plate containing stem cell‐specific medium. The number of spheroids (>100 μm) were counted. Representative photographs of spheroids (left panel), number of formed spheroids (right panel). Error bars represent mean ± SEM (n = 3). *P ≤0.05; **P ≤0.01.

Article Snippet: Transfection with OPG siRNA and universal scrambled sequence (30 nM) (Santa Cruz Biotechnology) was performed using the RNAiFect reagent (Qiagen) following the manufacturer recommendations.

Techniques: Transfection, Sequencing, Control, Western Blot, Staining, Cytometry