Journal: bioRxiv

Article Title: The circadian regulator PER1 inhibits osteoclastogenesis by activating inflammatory genes

doi: 10.1101/2025.09.18.677145

Figure Lengend Snippet: A. Relative expression levels of Per1 and Per2 in each cKO male osteoclasts in comparison to Cont osteoclasts on day 6 quantified with qRT-PCR. n = 3. B. Reconstructed 3D micro-CT images of femoral midshafts (top) and distal femurs (bottom) prepared from 12-week-old male mice of the indicated genotypes. Bar, 1 mm. C - G . Quantification of the cortical bone volume/total volume ratio ( C ), cortical thickness ( D ), trabecular bone volume/total volume ratio ( E ), trabecular number ( F ), and trabecular thickness ( G ) comparing 12-week-old male mice. n = 9 or 10. Mean ± SEM is shown. ** p < 0.01, * p < 0.05, and ns for not significant with two-way ANOVA with Tukey’s method of multiple comparisons.

Article Snippet: The cells were centrifuged, resuspended in Osteoclast Medium (Monocyte Medium with 100 ng/ml RANKL [Cell Signaling Technology, 68495]), and seeded at 8x10 5 cells/400 μl/well in a 48-well plate.

Techniques: Expressing, Comparison, Quantitative RT-PCR, Micro-CT

Journal: bioRxiv

Article Title: The circadian regulator PER1 inhibits osteoclastogenesis by activating inflammatory genes

doi: 10.1101/2025.09.18.677145

Figure Lengend Snippet: A. TRAP staining of the proximal tibial sections comparing Per1 cKO and Cont mice. Bar, 100 μm. B. Osteoclast numbers per bone perimeter in the proximal tibiae of the indicated genotypes. n = 5. C. Masson’s trichrome staining of the proximal tibial sections. Bar, 100 μm. D. Osteoblast numbers per bone perimeter in proximal tibiae of the indicated genotypes. n = 5. E. TRAP staining of osteoclasts in vitro on day 6. Bar, 500 μm. F. The numbers of osteoclasts per well in a 48-well plate on day 6. n = 5. G. Size distributions of osteoclasts on day 6. n = 3000 osteoclasts (600 osteoclasts/well x 5 wells of biologically independent experiments). H. Bone resorption assay stained with Toluidine blue. Bar, 2 mm. I. Areas of resorbed bones stained with Toluidine blue. n = 5. J. Histological sections and osteoclasts were prepared from 12-week-old male mice. Mean ± SEM is shown. ** p < 0.01, * p < 0.05, and ns for not significant with two-way ANOVA with Tukey’s method of multiple comparisons.

Article Snippet: The cells were centrifuged, resuspended in Osteoclast Medium (Monocyte Medium with 100 ng/ml RANKL [Cell Signaling Technology, 68495]), and seeded at 8x10 5 cells/400 μl/well in a 48-well plate.

Techniques: Staining, In Vitro

Journal: bioRxiv

Article Title: The circadian regulator PER1 inhibits osteoclastogenesis by activating inflammatory genes

doi: 10.1101/2025.09.18.677145

Figure Lengend Snippet: A. A volcano plot demonstrating differentially expressed genes between Per1 cKO and Cont osteoclasts. FC and padj indicate fold change and adjusted p value, respectively. B. A heatmap displaying up- or downregulated genes in Per1 cKO osteoclasts compared with Per1 Cont osteoclasts. C. Gene ontology analysis of downregulated genes in Per1 cKO osteoclasts compared with Per1 Cont osteoclasts. Pathways related to inflammation and immunity are underlined in red. D. A list of the genes that belong to the four pathways underlined in red in ( C ). Red and blue bars indicate Per1 cKO and Per2 cKO osteoclasts, respectively. E. A list of representative osteoclasts marker genes detected with RNA-seq. F. Relative expression levels of circadian regulators and osteoclasts marker genes in synchronized osteoclasts comparing three genotypes. The value of Per1 Cont cells at 24 hr was defined as 1.0 in each graph. Male osteoclasts were used in all data. ( A ) – ( E ) are based on biological triplicates, whereas ( F ) is based on biological triplicates with technical triplicated each. The red lines in ( D ) and ( E ) indicate FC = 1.5 or 0.67, and padj = 0.05. ** p < 0.01, * p < 0.05, and ns for not significant with the Cosinor analysis of circadian rhythmicity listed next to the gene names in color-coded manners in ( F ). ** p < 0.01 and * p < 0.05 with unpaired two-tailed t-test comparing peak levels of Per1 cKO and Cont osteoclasts embedded in each graph in ( F ).

Article Snippet: The cells were centrifuged, resuspended in Osteoclast Medium (Monocyte Medium with 100 ng/ml RANKL [Cell Signaling Technology, 68495]), and seeded at 8x10 5 cells/400 μl/well in a 48-well plate.

Techniques: Marker, RNA Sequencing, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: The circadian regulator PER1 inhibits osteoclastogenesis by activating inflammatory genes

doi: 10.1101/2025.09.18.677145

Figure Lengend Snippet: A. Relative expression levels of the indicated genes after KD with two independent siRNA sequences each. Data are based on biological triplicates with technical triplicates each. B. The numbers of osteoclasts in a well of a 48-well plate after KD of the indicated genes. n = 3. C. Size distributions of osteoclasts on day 6. n = 3000 osteoclasts (600 osteoclasts/well x 5 wells of biologically independent experiments). D. Concentrations of IL-1β in the osteoclast supernatant quantified with ELISA. n = 3. E. The numbers of osteoclasts in a well of a 48-well plate after culture with IL-1β. n = 5. F. Size distributions of osteoclasts after culture with IL-1β. n = 3000 (600 osteoclasts/per well x 5 wells of biologically independent experiments). All panels except for ( D ) used male Per1 Cont osteoclasts. Mean ± SEM is shown. ** p < 0.01, * p < 0.05, and ns for not significant with unpaired two-tailed t-test in comparison to control samples in ( A ) – ( D ) and two-way ANOVA with Tukey’s method of multiple comparisons in ( E ) and ( F ).

Article Snippet: The cells were centrifuged, resuspended in Osteoclast Medium (Monocyte Medium with 100 ng/ml RANKL [Cell Signaling Technology, 68495]), and seeded at 8x10 5 cells/400 μl/well in a 48-well plate.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison, Control

Journal: bioRxiv

Article Title: The circadian regulator PER1 inhibits osteoclastogenesis by activating inflammatory genes

doi: 10.1101/2025.09.18.677145

Figure Lengend Snippet: A. Relative expression levels of the indicated genes in synchronized osteoclasts comparing Per1 cKO, Per2 cKO, Per1 Cont osteoclasts. The value of Per1 Cont cells at 24 hr was defined as 1.0 in each graph. B. Relative expression levels of the indicated genes after overexpression of circadian regulators in RAW264.7 cells. Abbreviations are as follows. EV: empty vector and CB: Clock and Bmal1 . All experiments used male osteoclasts. Mean ± SEM is shown. ** p < 0.01, * p < 0.05, and ns for not significant with the Cosinor analysis of circadian rhythmicity listed next to the gene names in color-coded manners in ( A ). ** p < 0.01, * p < 0.05, and ns for not significant with unpaired two-tailed t-test comparing peak levels of Per1 cKO and Cont osteoclasts in ( A ) and with two-way ANOVA with Tukey’s method of multiple comparisons in ( B ).

Article Snippet: The cells were centrifuged, resuspended in Osteoclast Medium (Monocyte Medium with 100 ng/ml RANKL [Cell Signaling Technology, 68495]), and seeded at 8x10 5 cells/400 μl/well in a 48-well plate.

Techniques: Expressing, Over Expression, Plasmid Preparation, Two Tailed Test

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: Lipids

Article Title: Conjugated linoleic acid prevents ovariectomy-induced bone loss in mice by modulating both osteoclastogenesis and osteoblastogenesis

doi: 10.1007/s11745-013-3872-5

Figure Lengend Snippet: Eight weeks old C57BL/6 mice were ovariectomized or sham operated and fed experimental diets for 24 weeks. Thirty two weeks old mice were then sacrificed and splenocytes were isolated from spleen. (A) RNA was prepared, reverse transcribed and amplified by PCR using primers designed for genes of RANKL, OPG and GAPDH and visualized on agarose gels with ethidium bromide. (B) Relative expression of RANKL and OPG is shown. The intensity of the bands was determined by densitometry and normalized by the level of GAPDH. n=3 mice per group. P < 0.05 was considered significant. Flow cytometric analysis of RANKL expression in splenocytes (C, D and E). RANKL positive total splenocytes (C), RANKL positive CD8 T cells (D) and RANKL positive CD4 T cells (E) cells in splenocytes were analyzed for the expression of the cell surface determinants for RANKL only, CD8/RANKL and CD4/RANKL respectively. Before staining, FcγII/III receptors on cells were blocked by pre-incubation of the samples with a rat anti-CD32/16 Ab. Cells were then incubated with the anti-RANKL with or without anti-CD8 or anti-CD4 antibodies for 30 min at 4 °C. The cells were re-suspended in wash buffer and analyzed on a FACScan flow cytometer. n=3 mice per group. P value <0.05 was considered significant by student’s t test.

Article Snippet: Serum bone turnover markers measurement Bone resorption markers, receptor activator of NF-κB ligand (RANKL), and tartrate resistant acid phosphatase (TRAP)-5b and bone formation marker, N-terminal propeptide of type I procollagen (PINP) were measured in serum samples using mouse free soluble RANKL, mouse OPG, mouse TRAP5b, mouse PINP assay kits from Immunodiagnostic System (IDS) Inc. (Fountain Hills, AZ, USA) and bone formation marker, alkaline phosphatase (ALP) was measured in serum samples using Quantichrome alkaline phosphatase assay kit from Bioassay Systems (Hayward, CA, USA) according to the manufacturer’s instructions ( 17 ).

Techniques: Isolation, Reverse Transcription, Amplification, Expressing, Staining, Incubation, Flow Cytometry