Journal: The American journal of pathology

Article Title: Treatment with the matricellular protein CCN3 blocks and/or reverses fibrosis development in obesity with diabetic nephropathy.

doi: 10.1016/j.ajpath.2014.07.009

Figure Lengend Snippet: Figure 1 Stability of rhCCN3 in mouse plasma and the organ distribution of exogenously delivered rhCCN3 to C57JB mice in vivo. rhCCN3 was spiked into mouse plasma and incubated at 37C for 48 hours. Its stability was measured at different periods using an ELISA that recognizes intact CCN3 and differentiates the human form from endogenous mouse CCN3. A: rhCCN3 levels remained virtually unchanged during the 2-day incubation period. The dashed line indicates the average level of endogenous CCN3 as determined previously. B: rhCCN3 was injected by either the i.v. or i.p. route as a bolus at 50 times the circulating dose (938 ng/mL total), and plasma was collected 1, 8, 24, and 48 hours thereafter. Plasma or organ homogenates were assayed for rhCCN3. The results at 1 hour showed that in solid organs, rhCCN3 is easily measurable in the kidney, with much lower levels present in the liver and heart. C: Plasma levels were significantly greater at 1 hour than kidney levels, and the i.p. delivery mode produced the greatest levels in all areas sampled. Tissue and plasma samples taken at the later times produced no measurable CCN3 (data not shown), indicating that the approximate half-life for CCN3 detectable by this assay was 1 to 8 hours. Data are shown as means SEM. N Z 2 to 3, each run in triplicate.

Article Snippet: For mouse CCN3, the plate was first coated with rat monoclonal anti-mouse CCN3-specific trapping antibody (cat# MAB1976; R&D Systems, Minneapolis, MN).

Techniques: Clinical Proteomics, In Vivo, Incubation, Enzyme-linked Immunosorbent Assay, Injection, Produced

Journal: The American journal of pathology

Article Title: Treatment with the matricellular protein CCN3 blocks and/or reverses fibrosis development in obesity with diabetic nephropathy.

doi: 10.1016/j.ajpath.2014.07.009

Figure Lengend Snippet: Figure 9 Renal cortical laminin deposition and the effect of CCN3 treatment. Immunostaining for laminin in the renal cortex. AeD: Quali- tative analysis shows laminin deposition (dark brown) limited primarily to the glomerulus in nonobese/diabetic control mice (A); glomerular laminin deposition increased strongly in placebo-treated diabetic mice (B) and was greatly reduced in diabetic þ low-dose CCN3 (C) and diabetic þ high-dose CCN3 (D) mice. E: Quantitative results measuring laminin deposition as the percentage of glomerular area with marked deposition confirmed this up- regulation in disease and near-complete correction by rhCCN3. For the quantitative analysis, 25 glomeruli per animal were scored, and means were determined. Data are shown as means SEM of these means. N Z 8 to 9 animals per group. *P < 0.05. D, diabetic.

Article Snippet: For mouse CCN3, the plate was first coated with rat monoclonal anti-mouse CCN3-specific trapping antibody (cat# MAB1976; R&D Systems, Minneapolis, MN).

Techniques: Immunostaining, Control

Journal: The American journal of pathology

Article Title: Treatment with the matricellular protein CCN3 blocks and/or reverses fibrosis development in obesity with diabetic nephropathy.

doi: 10.1016/j.ajpath.2014.07.009

Figure Lengend Snippet: Figure 11 Glomerular hypertrophy, DN, and CCN3. Renal cortex sec- tions were stained by PASH and glomeruli were examined for size differ- ences at 17 weeks of age in healthy control mice (A) and placebo-treated obese/diabetic mice (B). C: Quantitation from image analysis (25 glomeruli per animal) was used to determine the mean glomerular size per group. The mean glomerular size increased approximately 15.3% in diabetes. This in- crease was reduced by approximately 50% on treatment with rhCCN3, and there seemed to be a dose-dependent effect. Data are shown as means SEM. N Z 8 to 9 mice per group. *P < 0.05 versus the placebo- treated diabetic group. D, diabetic.

Article Snippet: For mouse CCN3, the plate was first coated with rat monoclonal anti-mouse CCN3-specific trapping antibody (cat# MAB1976; R&D Systems, Minneapolis, MN).

Techniques: Staining, Control, Quantitation Assay

Journal: Nature

Article Title: A maternal brain hormone that builds bone.

doi: 10.1038/s41586-024-07634-3

Figure Lengend Snippet: Fig. 3 | Identification of CCN3 as a brain-derived osteoanabolic factor. a, Trabecular and cortical fractional BV, mechanical strength (three-point bend) and BMAT levels in long bones of Esr1fl/fl and Esr1Nkx2.1-cre females fed standard diet (SD) or HFD for 17 weeks (N = 4–6 per group). b, Representative images of tibia from female mice (aged 27 weeks) fed SD or HFD for 17 weeks labelled for calcein and Alizarin Red (top, white arrows and magnified from Extended Data Fig. 6c) and osmium stained with lipid droplets (bottom, yellow arrows). c, Heatmap of top DEGs changed in the ARC of Esr1Nkx2.1-cre females at 12 weeks of age (adapted from ref. 2) and at 27 weeks of age fed SD or HFD. Scale is log2 fold change. d, Transcript levels of Ccn3 and Penk in the ARC of 3.5-week-old mutant females, measured by quantitative PCR (qPCR). N = 2–3 per group. e, Ccn3 and Penk expression by RNAscope of the ARC in mutant female Esr1Nkx2.1cre mice fed

Article Snippet: CCN3 (R&D Systems, #AF1976 polyclonal goat, 1:1000 dilution).

Techniques: Derivative Assay, Staining, Mutagenesis, Real-time Polymerase Chain Reaction, Expressing, RNAscope

Journal: Nature

Article Title: A maternal brain hormone that builds bone.

doi: 10.1038/s41586-024-07634-3

Figure Lengend Snippet: Fig. 4 | CCN3 enhances osteogenesis, bone formation and fracture repair. a–c, Osteogenic differentiation assays (14 days). a, Differentiation of mouse ocSSCs (from 2-week-old male and female mice) treated with mCCN3, met- ENK and Bam22P. Inset, cells stained with Alizarin Red (Al Red) (n = 3). Veh, vehicle. b,c, Human ocSSCs treated with human CCN3 (hCCN3) during in vitro osteogenesis. b, Human ocSSCs from a 14-year-old male treated with hCCN3, met-ENK, Bam22P, gastric-related peptide (GRP) and follistatin (FST). c, Additional ocSCCs from 15-year-old (left), 72-year-old (middle) and 61-year- old (right) patients treated with hCCN3 (red bars) (n = 3), with representative images of wells stained with Alizarin Red to the right of the graph. F, female; M, male. d–f, Whole femur bone cultures treated with plasma or mCCN3 daily for 5 days. d, Left, %BV/TV for control Esr1fl/fl female (red) and male (blue) 6–8-week-old femurs treated with plasma from Esr1Nkx2.1-cre females; contralateral femurs treated with plasma from Esr1fl/fl mice (N = 19, 10). Right, per cent change in %BV/TV of contralateral female (red bar) or male (blue bar) femurs. e, Representative µCT images from treated femurs. f, Left, %BV/TV control

Article Snippet: CCN3 (R&D Systems, #AF1976 polyclonal goat, 1:1000 dilution).

Techniques: Staining, In Vitro, Clinical Proteomics, Control

Journal: Nature

Article Title: A maternal brain hormone that builds bone.

doi: 10.1038/s41586-024-07634-3

Figure Lengend Snippet: Fig. 5 | Exogenous CCN3 drives higher mass, strength and formation of bone in vivo. a, Left, schematic of experiment to induce loss-of-function of CCN3 (Ccn3 knockdown) in the ARC in mice. Right, Ccn3-positive neurons in female Esr1Nkx2.1-cre ARC versus %BV/TV after Ccn3 siRNA injections. b, Ccn3 expression in control, unilateral and bilateral hit with corresponding µCT scans of distal femurs. Scale bar, 500 µm (N = 6, 4). c, Left, schematic of experiment to induce gain-of-function CCN3 in the liver. Right, ectopic mCCN3 expression in Esr1fl/fl female hepatocytes following retro-orbital injection of AAVdj-CAG-CCN3 (AAVdj-CCN3) or control (AAVdj-Ctrl) vectors. Inset shows double nuclei. Scale bar, 100 µm. d, mCCN3 immunoblot of heparin–agarose-purified plasma (left) and liver extracts (right, 10 µg total protein) from mice 5 weeks after injection with AAVdj-Ctrl (–) or AAVdj-mCCN3 (+). Recombinant mCCN3 (rCCN3) shown in far left lane. e, %BV/TV (left) of femurs and L5 and mechanical strength (right) of femurs from 3-4-month-old Esr1fl/fl female mice 5 weeks after injection (N = 7, 8 femurs and N = 4, 3 L5). f, %BV/TV (left) of femurs and L5 and mechanical

Article Snippet: CCN3 (R&D Systems, #AF1976 polyclonal goat, 1:1000 dilution).

Techniques: In Vivo, Knockdown, Expressing, Control, Injection, Western Blot, Purification, Clinical Proteomics, Recombinant

Journal: Nature

Article Title: A maternal brain hormone that builds bone.

doi: 10.1038/s41586-024-07634-3

Figure Lengend Snippet: Fig. 6 | Lactating females require maternal brain CCN3 to sustain progeny. a, Representative images of coronal brain sections from Esr1fl/fl females stained for ERα (magenta) and CCN3 (green) in the posterior medial basal ARC during pregnancy and postpartum stages (lactation and post weaning) (N ≥ 2 for each time point). Scale bar, 50 µm. b, Colocalization of Ccn3 (green), Kiss1 (red) and Esr1 (magenta and cyan) transcripts in the ARC of a lactating control (Esr1fl/fl) female at 7 days. Scale bar, 50 µm. c, Relative Ccn3 quantified from microdissected ARC tissue obtained from Esr1fl/fl virgin, Esr1Nkx2.1-cre mutant virgin and Esr1fl/fl lactating (7 DPP) female mice (N = 5, 3, 3). d, CCN3 (green) and vimentin (red, VIM) in the posterior ARC of virgin Esr1Nkx2.1-cre mutant and lactating or OVX Esr1fl/fl control mice (1 week after surgery). e–i, Ccn3 knockdown (KD) in the ARC of lactating female mice. e, Schematic of injection of shRNA Ccn3 (shCcn3) or shRNA control (shCtrl) vectors into the ARC of control Esr1fl/fl females with experimental timeline (left) and representative images of CCN3 at 12 DPP (right). Scale bar, 500 µm. LCD, low-calcium diet. f,g, Litter sizes (f) and

Article Snippet: CCN3 (R&D Systems, #AF1976 polyclonal goat, 1:1000 dilution).

Techniques: Staining, Control, Mutagenesis, Knockdown, Injection, shRNA