Journal: iScience

Article Title: A novel macrolide–Del-1 axis to regenerate bone in old age

doi: 10.1016/j.isci.2024.108798

Figure Lengend Snippet: Local treatment with DEL-1-Fc promotes bone regeneration in 12-month-old mice (A) Experimental design. (B, left) Measurement of bone heights (distance from cement-enamel junction [CEJ] to alveolar bone crest [ABC]; CEJ-ABC) in groups of 2-month-old and 12-month-old mice after ten days of ligature placement (10dL) or after 10 days of ligature placement followed by five days without ligatures to enable resolution from periodontitis (10dL + 5dR). (B, middle) Data from the left panel were transformed to show bone loss at ligated (L) sites vs. unligated (U) contralateral sites. (B, right) Data from the middle panel were transformed to show bone growth relative to corresponding 10dL group (baseline). (C) Experimental design. (D) 12-month-old mice were subjected to LIP for ten days with or without ligature removal for five days to enable resolution, with or without local microinjection with DEL-1-Fc (1 μg) or equal molar amount of Fc control. Treatments were performed daily on days 10–14. (D, left) Measurement of bone heights (CEJ-ABC distance). (D, middle) Data from the left panel were transformed to show bone loss in ligated (L) sites vs. unligated (U) contralateral sites. (D, right) Data from the middle panel were transformed to indicate bone growth relative to the 10dL group (baseline). Data are means ± S.D. (B, n = 5–6 mice/group; D, n = 4–7 mice/group). ∗p < 0.05, ∗∗p < 0.01; one-way ANOVA and Dunnett’s test.

Article Snippet: Rabbit monoclonal anti-DEL-1 (clone EPR12451) , Abcam , Cat# ab190692.

Techniques: Transformation Assay

Journal: iScience

Article Title: A novel macrolide–Del-1 axis to regenerate bone in old age

doi: 10.1016/j.isci.2024.108798

Figure Lengend Snippet: Macrolides induce bone regeneration in a DEL-1-dependent manner in 18-month-old mice with naturally occurring periodontitis (A and B) Aged WT and aged Del1 −/− mice were administered macrolide antibiotics or vehicle control (ethanol) intraperitoneally daily for nine days. (C) Aged WT were administered macrolide antibiotics intraperitoneally twice a week for eight weeks. (A–C) The distance from the CEJ-ABC was measured (left panels). The CEJ-ABC data were transformed to show the bone changes relative to the control, which was set as the baseline (right panels). Positive values (in mm) indicate bone growth relative to the baseline (control). (D and E) Analysis of gene expression from gingiva samples collected from (D) aged WT and (E) aged Del1 −/− mice after nine days of drug treatment. The mRNA levels of the indicated genes were quantified by qPCR. Data were normalized to Gapdh mRNA and plotted relative to the aged control group, set as 1. (F and G) Coronal maxillary sections from (F) aged WT and (G) aged Del1 −/− mice were stained with modified Masson’s trichrome stain, which stains the mature (old) bone blue and new bone formation red . T: tooth; PDL: periodontal ligament; OB: old bone as blue; NB : new bone as red . Scale bars, 100 μm. Data are means ± SD (A, n = 8 mice/group; B, n = 4–6 mice/group; C, n = 6 mice/group; D-E, n = 5 mice/group). ∗∗p < 0.01; one-way ANOVA and Dunnett’s test.

Article Snippet: Rabbit monoclonal anti-DEL-1 (clone EPR12451) , Abcam , Cat# ab190692.

Techniques: Transformation Assay, Expressing, Staining, Modification

Journal: iScience

Article Title: A novel macrolide–Del-1 axis to regenerate bone in old age

doi: 10.1016/j.isci.2024.108798

Figure Lengend Snippet: Macrolides induce DEL-1 protein expression in PDL spaces of the aged periodontium and promote the binding of C/EBPβ to the DEL1 promoter Aged WT and aged Del1 −/− mice were administered macrolide antibiotics or vehicle control (ethanol) intraperitoneally daily for nine days. (A) Representative IF images of maxillary sections from aged WT mice obtained with fluorescence microscopy. Frozen maxillae sections were stained for DEL-1 (green), CD31 (red), and the nuclei were counterstained using DAPI (blue). T: Tooth; PDL: periodontal ligament; B: alveolar bone. Scale bars, 100 μm. (B) Mean fluorescence intensity (MFI) values were measured from randomly selected IF images from aged WT mice using the ImageJ software. All MFI values were normalized and plotted relative to the MFI of the control group, set as 1. (C) Representative IF images of maxillary sections from aged Del1 −/− mice obtained with fluorescence microscopy. Frozen maxillae sections were stained for DEL-1 (green), CD31 (red), and the nuclei were counterstained using DAPI (blue). T: tooth; PDL: periodontal ligament; B: alveolar bone. Scale bars, 100 μm. (D) The mRNA levels of DEL1 from hPDLCs were quantified by qPCR on day seven of the drug administration. Data were normalized to GAPDH mRNA and plotted relative to the aged control group, set as 1. (E) hPDLCs were transiently transfected with hEDIL3-promoter-Luc reporter plasmid, pre-treated for 30 min with ethanol control, or indicated macrolides (10 μg/mL), and analyzed for luciferase activity. A renilla luciferase construct was co-transfected as an internal control for normalization. Data are calculated as fold change relative to ethanol control treatment, which was set as 1. (F) ChIP analysis of C/EBPβ binding capacity at the DEL1 promoter in hPDLCs treated for 4 h with ethanol control or indicated macrolides (10 μg/mL). Non-immunoprecipitated cell extracts were used as input samples. Data are plotted as a percentage of input. Data are means ± SD (B, n = 6 mice/group; D, n = 3 sets of cultures/group; E, n = 6 sets of cultures/group; F, n = 4 sets of cultures/group). ∗p < 0.05, ∗∗p < 0.01; one-way ANOVA and Dunnett’s test.

Article Snippet: Rabbit monoclonal anti-DEL-1 (clone EPR12451) , Abcam , Cat# ab190692.

Techniques: Expressing, Binding Assay, Fluorescence, Microscopy, Staining, Software, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Construct, Immunoprecipitation

Journal: iScience

Article Title: A novel macrolide–Del-1 axis to regenerate bone in old age

doi: 10.1016/j.isci.2024.108798

Figure Lengend Snippet: EM-523, a non-antibiotic erythromycin derivative, promotes bone regeneration in aged mice (A and B) Aged WT mice were treated with ethanol control, ERM, or EM-523 intraperitoneally (A) daily for nine days or (B) twice a week for eight weeks. The distance from the CEJ-ABC was measured (left panels). The CEJ-ABC data were transformed to show the bone changes relative to the control, which was set as the baseline (right panels). Positive values (in mm) indicate bone growth relative to the baseline (control). (C) Representative IF images of maxillary sections obtained by fluorescence microscopy. Frozen maxillae sections were stained for DEL-1 (green), CD31 (red), and the nuclei were counterstained using DAPI (blue). T: tooth; PDL: periodontal ligament; B: alveolar bone. Scale bars, 100 μm. (D) Mean fluorescence intensity (MFI) values of DEL-1 were measured from randomly selected IF images from aged WT using the ImageJ software. All MFI values were normalized and presented relative to the MFI of the control group in aged WT mice, set as 1. (E) The mRNA levels of DEL1 from human periodontal ligament cells (hPDLCs) were quantified by qPCR on day seven after drug treatment. Data were normalized to GAPDH mRNA and plotted relative to the aged control group, set as 1. (F) hPDLCs were transiently transfected with hDEL1-promoter-Luc reporter plasmid, pre-treated for 30 min with ethanol control, ERM (10 μg/mL), or EM-523 (1 μg/mL), and analyzed for luciferase activity. A renilla luciferase construct was co-transfected as an internal control for normalization. Data were calculated as fold change relative to ethanol control treatment, which was set as 1. (G) ChIP analysis of C/EBPβ binding capacity at the DEL1 promoter in hPDLCs treated for 4 h with ethanol control, ERM, or EM-523 (10 and 1 μg/mL, respectively). Non-immunoprecipitated cell extracts were used as input samples. Data are plotted as a percentage of input. (H) Gene expression analysis using qPCR of primary mouse PDLCs on day nine of the osteogenic differentiation assay for the expression of the indicated genes. Cells were treated with vehicle control, macrolides, or EM-523 (ERM, CLR and AZM as 10 μg/mL; EM-523 as 1 μg/mL). Data were normalized to Gapdh mRNA and plotted relative to ethanol-treated control, set as 1. (I–N) Aged WT mice were treated with ethanol control, ERM, or EM-523 intraperitoneally for nine days. (I) Optical microscopy images of WT maxillary sections show PDL areas stained for ALP activity. T: Tooth; PDL: periodontal ligament; B: alveolar bone. Scale bars, 100 μm. (J) The ALP-positive region of each area was quantified and represented as a percentage of the total area. (K) Optical microscopy images of WT maxillary sections show PDL areas stained for TRAP. Arrowheads indicate TRAP+ cells in PDL space. T: Tooth; PDL: periodontal ligament; B: alveolar bone. Scale bars, 100 μm. (L) The number of TRAP + cells within the PDL space were counted and compared between indicated groups. (M) Representative IF images of maxillary sections obtained by fluorescence microscopy. Frozen maxillary sections were stained for alpha-smooth muscle actin (α-SMA, green), and the nuclei were counterstained using DAPI (blue). T: tooth; PDL: periodontal ligament; B: alveolar bone. Scale bars, 100 μm. (N) Mean fluorescence intensity (MFI) values were measured from IF images using the ImageJ software. All MFI values were normalized and plotted relative to the MFI of the control group in aged mice, set as 1. (O) Cultures of hPDLCs were treated with ethanol control, ERM, or EM-523 (10 and 1 μg/mL). The mRNA levels of ACTA2 from hPDLCs were quantified by qPCR on day seven after drug treatment. Data were normalized to GAPDH mRNA and plotted relative to the aged control group, set as 1. Data are means ± S.D. (A, n = 8–10 mice/group; B, n = 6 mice/group; D, n = 6 mice/group; E, n = 3 sets of cultures/group; F, n = 6 sets of cultures/group; G, n = 4 sets of cultures/group; H, n = 3–4 sets of cultures/group; J, n = 6 mice/group; L, n = 9 mice/group; N, n = 6 mice/group; O, n = 5 sets of cultures/group). ∗p < 0.05, ∗∗p < 0.01; (one-way ANOVA and Tukey’s test; only H, one-way ANOVA and Bonferroni’s test).

Article Snippet: Rabbit monoclonal anti-DEL-1 (clone EPR12451) , Abcam , Cat# ab190692.

Techniques: Transformation Assay, Fluorescence, Microscopy, Staining, Software, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Construct, Binding Assay, Immunoprecipitation, Expressing, Differentiation Assay

Journal: iScience

Article Title: A novel macrolide–Del-1 axis to regenerate bone in old age

doi: 10.1016/j.isci.2024.108798

Figure Lengend Snippet: EM-523 promotes bone regeneration and suppresses osteoclastogenesis in vitro (A) MC3T3-E1 cells were cultured in osteogenic differentiation medium treated with control solvent or indicated macrolides. (A, left panel) Representative images of mineralization nodules from osteoblasts, stained with Alizarin Red S (ARS) staining after 26 days of the osteogenic differentiation assay. (A, middle panel) The mineralization area in each culture was quantified and represented as a percentage of the total area. (A, right panel) Quantification of the ARS staining using 10% cetylpyridinium chloride (CPC) on day 26 of the osteogenic differentiation assay. The absorbance was measured at 560 nm. The control group was incubated with an osteogenic differentiation medium only. (B) MC3T3-E1 cells were cultured in osteogenic differentiation medium treated with ethanol, ERM, or EM-523. (B, left panel) Representative images of mineralization nodules from osteoblasts, stained with ARS after 20 days of the osteogenic differentiation assay. (B, right panel) The mineralization area in each culture was quantified and presented as a percentage of the total area. The control group was incubated with an osteogenic differentiation medium only. (C) hPDLCs were cultured in osteogenic differentiation medium with control solvent or indicated macrolides. (C, left panel) Representative images of hPDLCs cultures, stained with alkaline phosphatase stain after 26 days of the osteogenic differentiation assay. Scale bars, 200 μm. (C, right panel) The ALP-positive region of each culture was quantified and represented as a percentage of the total area. (D) Gene expression analysis of hPDLCs on day nine of the osteogenic differentiation assay using qPCR. RUNX2 , SP7 , and BGLAP , as representative markers for early, middle, and late osteogenic markers, respectively. Data were normalized to GAPDH mRNA and plotted relative to ethanol-treated control, set as 1. (E) hMSCs were cultured in osteogenic differentiation medium with ethanol or indicated macrolides (10 μg/mL). (E, upper panel) Representative images of mineralization nodules from osteoblasts, stained with Alizarin Red S after 26 days of the osteogenic differentiation assay. (E, middle left panel) The mineralization area in each culture was quantified and presented as a percentage of the total area. (E, middle right panel) Quantification of the Alizarin Red S staining using 10% CPC on day 26 of the osteogenic differentiation assay. (E, lower panel) Gene expression analysis using qPCR of human MSCs on day nine of the osteogenic differentiation assay for the expression of the indicated genes. Data were normalized to GAPDH mRNA and plotted relative to ethanol control, set as 1. (F) Bone marrow cells were collected from WT mice for the osteoclastogenesis assay. Cells were treated with DEL-1-Fc (1 μg/mL), equal molar amount of Fc control, control solvent, or indicated macrolides for one week. (F, upper panel) Representative images of the culture well from each group after TRAP staining are shown. Histological images of TRAP + MNCs. Scale bars, 200 μm. (F, lower left panel) The average size of TRAP + MNCs was measured using the ImageJ software. (F, lower right panel) The percentage of TRAP + area per total area was measured using the ImageJ software. (G) Primary osteoblastic progenitor cells were collected from Del1 −/− mice. Primary osteoblastic progenitor cells were cultured in osteogenic differentiation media administered with DEL-1-Fc, equal molar amount of Fc control, control solvent, or indicated macrolides for 26 days. (G, upper panel) Representative images of mineralization nodules from osteoblasts, stained with Alizarin Red S after 26 days of the osteogenic differentiation assay. (G, lower panel) Gene expression analysis was completed on day nine of the osteogenic differentiation assay using qPCR. Runx2 , Sp7 , and Bglap , as representative markers for early, middle, and late osteogenic markers, respectively. Data were normalized to GAPDH mRNA and plotted relative to ethanol control, set as 1. (H) Bone marrow cells were collected from Del1 −/− mice for the osteoclastogenesis assay. Cells were treated with DEL-1-Fc, equal molar amount of Fc control, control solvent, or indicated macrolides for one week. (H, upper panel) Representative images of the culture well from each group after TRAP staining were shown. Histological images of TRAP + MNCs. Scale bars, 200 μm. (H, lower left panel) The average size of TRAP + MNCs was measured using the ImageJ software. (H, lower right panel) The percentage of TRAP + area per total area was measured using the ImageJ software. Data are means ± S.D. (A–C, n = 4 sets of cultures/group; D, n = 5 sets of cultures/group; E middle and lower panel, n = 4 sets of cultures/group; F–H, n = 5 sets of cultures/group). ∗p < 0.05, ∗∗p < 0.01; one-way ANOVA and Bonferroni’s test.

Article Snippet: Rabbit monoclonal anti-DEL-1 (clone EPR12451) , Abcam , Cat# ab190692.

Techniques: In Vitro, Cell Culture, Solvent, Staining, Differentiation Assay, Incubation, Expressing, Software

Journal: iScience

Article Title: A novel macrolide–Del-1 axis to regenerate bone in old age

doi: 10.1016/j.isci.2024.108798

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-DEL-1 (clone EPR12451) , Abcam , Cat# ab190692.

Techniques: Recombinant, Construct, Transfection, Staining, Chromatin Immunoprecipitation, Magnetic Beads, Luciferase, SYBR Green Assay, Generated, Binding Assay, Expressing, Plasmid Preparation, Software, Imaging, Microscopy, Fluorescence, Real-time Polymerase Chain Reaction