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