Journal: The International Journal of Biochemistry & Cell Biology

Article Title: Apolipoprotein(a) acts as a chemorepellent to human vascular smooth muscle cells via integrin α V β 3 and RhoA/ROCK-mediated mechanisms ☆

doi: 10.1016/j.biocel.2013.05.021

Figure Lengend Snippet: Mechanism of apo(a)-induced effects. (A) SV-SMC were transfected with 25 nM RhoA siRNA; knockdown was confirmed via immunoblotting. Numbers represent densitometry data for RhoA ( n = 6). (B) Number of migrated cells per HP field under basal conditions and in response to apo(a) following RhoA silencing (closed bars) compared to mock transfected cells (open bars, n = 5). (C). Migration under basal conditions and in response to apo(a) following pre-treatment with vehicle control (0.002% dH 2 O; open bars) or 10 μM ROCK inhibitor, Y-27632 (closed bars) for 1 h ( n = 6). (D) Migration in response to apo(a) following pre-treatment with vehicle control (0.001% dH 2 O; open bars) or 1 μM fluvastatin (closed bars) for 2 h ( n = 5). (E). Migration in response to apo(a) following pre-treatment with vehicle control (0.001% PBS; open bars) or 5 μg/mL αvβ 3 integrin neutralising antibody (closed bars) for 5 min ( n = 5). (F) Migration in response to apo(a) following pre-treatment with vehicle control (0.001% DMSO; open bars) or 100 μM genistein (closed bars) for 1 h ( n = 5). *** P < 0.001, ** P < 0.01, * P < 0.05, NS = non-significant.

Article Snippet: Neutralising antibodies against TGFβRII (AF-241-NA) and integrin α V β 3 (MAB1976 clone LM109) were from R&D Systems and Millipore respectively.

Techniques: Transfection, Knockdown, Western Blot, Migration, Control

Journal: The International Journal of Biochemistry & Cell Biology

Article Title: Apolipoprotein(a) acts as a chemorepellent to human vascular smooth muscle cells via integrin α V β 3 and RhoA/ROCK-mediated mechanisms ☆

doi: 10.1016/j.biocel.2013.05.021

Figure Lengend Snippet: Summary figure. Our data suggest that apo(a) interacts with integrin α V β 3 on the surface of vascular SMC. This signals through tyrosine kinases to activate RhoA and induce stress fibre formation, cell spreading and chemorepulsion. These molecular effects may have a negative impact on smooth muscle cell remodelling contributing towards atherosclerosis, vessel stiffness, and impaired vein graft integration and arterialisation.

Article Snippet: Neutralising antibodies against TGFβRII (AF-241-NA) and integrin α V β 3 (MAB1976 clone LM109) were from R&D Systems and Millipore respectively.

Techniques:

Journal: bioRxiv

Article Title: Brain-Derived CCN3 Is An Osteoanabolic Hormone That Sustains Bone in Lactating Females

doi: 10.1101/2023.08.28.554707

Figure Lengend Snippet: a , Fractional bone volume, mechanical strength, and BMAT levels in long bones of Esr1 fl/fl and Esr1 Nkx2.1-Cre females fed SD or HFD for 17 weeks (N = 4-6 per group). b , Representative images of tibia stained for TRAP (top row), labeled for Calcein/ Alizarin Red (middle row) and osmium stained (bottom row); double labeling (white arrows), lipid droplets (yellow arrows). c , Normalized reads for candidate genes; Penk in the pituitary (N = 2-4). d , Heatmap of top DEGs changed in Esr1 N-kx2.1-Cre females at 12 weeks of age (adapted from RE), and at 27 weeks of age fed SD or HFD; Scale is Log2 fold. e , Transcript levels of Ccn3 and Penk in mutant female ARC by qPCR. f , Staining for ERa (pink) and CCN3 (green) in brain sections from posterior ARC and SCN regions of Esr1 fl/fl female (10 wks) and Esr1 Nkx2.1-Cre female and male (12 wks), Scale bar = 100 and 200 μm. g , Merge of CCN3 and KISS1 expression in Esr1 Nkx2.1-Cre female ARC. One-way ANOVA in panels a and c (Šidák’s multiple-comparisons test). Unpaired Student’s T-test, 2-tailed for panels e. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Error Bars ± SEM. Abbreviations: ME median eminence, ARC arcuate nucleus, 3V third ventricle, SCN suprachiasmatic nucleus, oc optic chiasm.

Article Snippet: Immunohistochemistry was performed using primary antibodies against ERa (EMD Millipore, #C1355 polyclonal rabbit, 1:750 dilution) and CCN3 (R&D Systems, #AF1976 polyclonal goat, 1:1000 dilution) diluted in PBS with 0.1% Triton-X100, 5% normal donkey serum, and 5% BSA.

Techniques: Staining, Labeling, Mutagenesis, Expressing

Journal: bioRxiv

Article Title: Brain-Derived CCN3 Is An Osteoanabolic Hormone That Sustains Bone in Lactating Females

doi: 10.1101/2023.08.28.554707

Figure Lengend Snippet: a , Fractional bone volume for control Esr1 fl/fl female (red circles) and male (blue circles) femurs cultured ex vivo and treated daily for 5 days with plasma isolated from Esr1 Nkx2.1-Cre mutant females (6-12 wks); contralateral femurs were treated with control plasma (white circles), (N = 19, 10). b , Data from panel a, showing percent change in bone volume of contralateral female (red bar) or male (blue bar) femurs after adding mutant versus control plasma. c , Representative μCT images scanned from female and male femurs treated with plasma with corresponding %BV/TV. d , Fractional bone volume of female (N =11) and male (N=8) control femurs treated daily with recombinant mouse CCN3 (mCCN3, 0.3 nM) compared to untreated baseline control (Baseline). e , Percent change in bone volume of female (red bar) or male (blue bar) femurs treated with either CCN3 or saline and then compared to the baseline value of freshly isolated and stored contralateral femur (N =5-10). f , Representative images of H&E-stained femurs at baseline and after culturing with CCN3. g , Percent change in %BV/TV and trabecular thickness in Esr1 fl/fl females and males following daily CCN3 injections (i.p. 7.5 μg/kg) or saline for 21 days. All data were normalized to the average for control females injected with only saline (N = 6,8 for females and 7,6 for males). h , Representative μCT images scanned from female and male treated femurs with corresponding %BV/TV. i , Osteogenic differentiation assays of purified mouse ocSSCs treated with recombinant mouse (m)CCN3 protein or the Penk-encoded met-ENK and Bam22P peptides. Plotted values are normalized to Alizarin staining of control wells in defined media alone, set at 100% with brightfield images of wells below without or with mCCN3 (n = 3 per condition). j , Osteogenic differentiation assays of purified human ocSSCs treated with recombinant human (h)CCN3 protein. Plotted values are normalized to Alizarin staining of control wells in defined media alone set at 100%, and representative images of wells treated without or with mCCN3 below (n = 3 per condition). One-way ANOVA in panels e, k, and i (Šidák’s multiple-comparisons test). Paired Student’s T-test, 2-tailed for panels a, d and Unpaired Student’s T-test for panels b, g. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Error Bars ± SEM.

Article Snippet: Immunohistochemistry was performed using primary antibodies against ERa (EMD Millipore, #C1355 polyclonal rabbit, 1:750 dilution) and CCN3 (R&D Systems, #AF1976 polyclonal goat, 1:1000 dilution) diluted in PBS with 0.1% Triton-X100, 5% normal donkey serum, and 5% BSA.

Techniques: Control, Cell Culture, Ex Vivo, Clinical Proteomics, Isolation, Mutagenesis, Recombinant, Saline, Staining, Injection, Purification

Journal: bioRxiv

Article Title: Brain-Derived CCN3 Is An Osteoanabolic Hormone That Sustains Bone in Lactating Females

doi: 10.1101/2023.08.28.554707

Figure Lengend Snippet: a , Ectopic expression of mCCN3 in hepatocytes with their characteristic double nuclei following retroorbital injection of AAVdj-CAG-CCN3 compared to injections of control AAVdj viral vector (AA-Vdj-Ctrl), Scale bar = 100 μm. b , Representative images of femur sections stained with Alizarin Red and magnified area of double-labeled bone surfaces obtained from control females injected with 100 μL of low (5*10 GC/mL) or high (3*10 GC/mL) AAVdj-CAG-CCN3 dose of viral vector. c , BV/TV (%) from μCT and bone formation rate (BFR) plotted as determined by dynamic histomorphometry analyses and assessed by Calcein (green) and Alizarin Red (red). Low dose (N = 4,6, High Dose N = 7,8). d , Injection of siRNA directed against mCcn3 into ARC of mutant Esr1 Nkx2 . 1-Cre mutant females. e , Ccn3 expression in posterior ARC shown for scramble control oligos, a unilateral hit, and the best level of knockdown. Scale bar = 100 μm. Lower panels show corresponding μCT scans of sagittal and longitudinal views of distal femur; complete misses were added to the control group (N = 4, 4). f , XY plot of the number of CCN3-postivie neurons in the posterior ARC versus the fractional bone volume. g , Representative images of coronal female brain sections stained for ERα (pink) and CCN3 (green) in the posterior ARC region during pregnancy or different postpartum stages as indi-cated; higher resolution images are shown in the lower row (N ≥ 2). Scale bar = 100 μm upper row, 50 μm lower row. h , Ccn3 transcripts quantified from microdissected ARC tissue obtained from from Esr1 fl/fl virgin, Esr1 Nkx2 . 1-Cre virgin, and Esr1 fl/fl lactating (7DPP) females (N = 5, 3, 3) . I , Schematic of brain-derived CCN3 as an osteoanabolic hormone that counteracts the catabolic actions of mammary-gland PTHrP to maintain adequate calcium and preserve the maternal skeleton during lactation when E2 levels drop. One-Way ANOVA for panel g (Šidák’s multiple-comparisons test), Unpaired Student’s T-test for panels b. *p < 0.05, **p < 0.01, ****p < 0.0001, ns = not significant. Error Bars ± SEM. Abbreviations: ME median eminence, ARC arcuate nucleus, 3V third ventricle.

Article Snippet: Immunohistochemistry was performed using primary antibodies against ERa (EMD Millipore, #C1355 polyclonal rabbit, 1:750 dilution) and CCN3 (R&D Systems, #AF1976 polyclonal goat, 1:1000 dilution) diluted in PBS with 0.1% Triton-X100, 5% normal donkey serum, and 5% BSA.

Techniques: Expressing, Injection, Control, Plasmid Preparation, Staining, Labeling, Mutagenesis, Knockdown, Derivative Assay