CCN3 modifies the expression of several genes involved in the control of cell cycle progression.

<p>A) mRNA levels of cells treated with or without CCN3 for 24 h. B) Reported changes in gene expression following CCN3 treatment. Results represent means +/– SEM of 3 separate experiments. *, p<0.05; **, p<0.01; ns  =  not significant.</p

CCN3 impairs glucose-stimulated insulin secretion.

<p>A) We evaluated the effects of <i>Ccn3</i> overexpression on insulin secretion by the hGH co-transfection system. In brief, we measured the hGH levels in the media of INS832/13 cells co-transduced with hGH and CCN3 or the empty plasmid and incubated at 2.8 mM (G2.8) or 16 mM glucose (G16). KCl (35 mM) was used as control. B) We studied the effects of CCN3 protein on insulin secretion. Insulin released in the culture medium was measured following incubation of INS832/13 cells in 2.8 mM or 16 mM glucose/KRBH medium or in the presence of 35 mM KCl. Secreted insulin levels were normalized to protein content. C) Measurements of intracellular calcium in living INS832/13 cells treated as described in (B) using the Fura-2 dye. We measured the ratio of fluorescence signals produced by excitation of 340 nm and 380 nm and detected at 510 nm to determine intracellular calcium concentrations. D) Glucose oxidation was measured as ¹⁴CO₂ production from [U-¹⁴C]-glucose in cells treated as described in (A) and the results were normalized to protein content. E) Cellular insulin content was measured by Elisa after lysis of the cells in acidic ethanol. Results represent mean ± SEM of three separate experiments carried out in triplicate. *, p<0.05.</p

CCN3 is up-regulated in animal models of insulin resistance.

<p>A) We determined CCN3 protein levels in different genetic models of insulin resistance. We prepared paraffin sections from WT, CAFoxO1 transgenics (305), <i>db/db</i>, and <i>Irs2^–/–</i> mice and performed CCN3 immunostaining (at least n = 3 for each). Representative images are shown. B) <i>Ccn3</i> expression in both the exocrine and the endocrine fraction was determined by PCR in 2 months old male animals. Amylase and insulin were used as specific exocrine and endocrine markers. Actin was used as a control in each sample. Representative images of 3 separate experiments are shown.</p

CCN3 protein decreases β-cell proliferation.

<p>A) The effect of CCN3 protein (1 nM) on β-cell proliferation was evaluated by BrdU incorporation in INS832/13 cells incubated at 5 mM glucose (G5), 25 mM glucose (G25) or in the presence of 10% serum. B) We studied the effects of 1 nM CCN3 protein on cAMP levels in INS cells by ELISA. C–D) Dose-dependent effects of CCN3 on cAMP levels in isolated rat islets (C) and INS cells (D). E) We measured <i>Ccn3</i> expression in cells transduced with increasing concentrations of siRNA specifically targeting <i>Ccn3</i> mRNA or scrambled siRNA as control by qPCR. F) Proliferation of INS832/13 cells transduced with either 50 pmol CCN3 or control siRNA. Results represent mean ± SEM of three separate experiments carried out at least in duplicate. *, p<0.05; **, p<0.01.</p

<i>Ccn3</i> is a transcriptional target of FoxO1 in β-cells.

<p>A) <i>Ccn3</i> expression by quantitative real-time qPCR in INS832/13 cells transduced with either Ad-β-Gal or Ad-CN-FoxO1 and cultured for 24 h. Results are means +/– SEM of 4 separate experiments. B) <i>Ccn3 </i>mRNA levels in INS832/13 cells treated with or without 10% serum and LY294002 (50 µM) for 4 h. Results represent means +/– SEM of 3 separate experiments. C) <i>Ccn3</i> expression in isolated islets from WT and transgenic mice with CAFoxO1 overexpression in their β-cells (called “305 mice”) (n = 5 for each). *, p<0.05.</p

β INS832/13 cells secrete CCN3 protein.

<p>A–B) Western blot of CCN3 proteins in the media (A) or whole cell extracts (B) of either AdGFP or AdCCN3 infected cells and control uninfected cells. In (B) the top arrow indicate full-length CCN3 at a molecular weight of 47 KDa, whereas the bottom arrow indicate a CCN3 fragment of 35 KDa that have both been described in the literature. C) Immunohistochemical analysis of endogenous CCN3 protein localization in INS832/13 cells. We performed triple immunohistochemistry with CCN3 (blue), insulin (green) and Vamp/synaptobrevin (red) antibodies. D) Simple immunohistochemistry for CCN3 combined with DAPI staining demonstrate both nuclear and cytoplasmic localization of CCN3 in untransfected cells (Control, top) and cells transfected with a scrambled siRNAs (SiCont, middle). No CCN3 expression could be detected in cells transfected with Ccn3 siRNAs after 48 h (SiCcn3, bottom). Representative images of 3 separate experiments are shown.</p

Modulation of the inflammatory response in Ara-C treated SOD1^G93A mice.

<p>Quantitative RT-PCR results (values are normalized to GADPH and relative to wild-type control treated with vehicle; no significant difference was found between wild-type controls treated with vehicle or M-CSF for any marker). Significant differences were found between Ara-C and vehicle treated SOD1^G93A in levels of mRNA for TGF-b1 (*<i>p</i> = 0.0148), IL-1b (**<i>p</i> = 0.0072), IL-6 (**<i>p</i> = 0.0014) and IGF-1 (*<i>p</i> = 0.0108). Note that all mRNA levels were significantly higher in vehicle treated transgenic mice compared to WT, treated or not (<i>p</i><0.0025), except for IL-4 (<i>p</i> = 0.7582). All values are mean ± SEM; <i>n</i>(WT) = 9, <i>n</i>(Tg-Vehicle) = 6, <i>n</i>(Tg-Ara-C) = 15.</p

Ablation of proliferating cells in CNS decreases lifespan of SOD1^G93A mice.

<p>Kaplan-Meier survival curve shows that transgenic mice Sod1^G93A (n = 7) treated with Ara-C between 75 and 115 days had a mean survival of 134 days while untreated SOD1^G93A (n = 9) had a mean survival of 141 days. Log-rank test shows that this difference is significant (<i>p</i> = 0.0081).</p

Ara-C treatment caused a decrease in microglia, NG2+ progenitors, oligodendrocytes, astrocytes and T cells in the spinal cord of SOD1^G93A mice.

<p>Immunofluorescence for cell markers Iba1 (A), CD68 (B), NG2 (C), GFAP (D), CD3 (E) and Olig2 (F) in the lumbar spinal cord of mutant SOD1 transgenic mice treated with vehicle or Ara-C. (G) Quantification of Iba positive cells showed a 1.5 folds reduction of cells in Ara-C treated mice compared to controls (**<i>p</i> = 0.0086). (H) Quantification of CD68 marker showed a 1.5 folds reduction of cells in Ara-C treated mice compared to controls (**<i>p</i> = 0.0099). (I) Quantification of NG2+ marker showed a 1.7 folds reduction of cells in Ara-C treated mice compared to controls (<i>p</i> = 0.0713) (J) Quantification of GFAP marked cells showed a slightly reduced number of astrocytes (1.2 folds) in Ara-C treated mice compared to controls, although this result was not significant (<i>p</i> = 0.3430). (K) Quantification of Olig2 positive cells showed a 2.0 folds reduction of cells in Ara-C treated mice compared to controls (*<i>p</i> = 0.0236) (L) Quantification of CD3+ cells showed 3.8 folds reduction of cells in Ara-C treated mice compared to controls (*<i>p</i> = 0.0122). All mice were analyzed at 115 days. All values are means ± SEM. Scale bars: 100 µm.</p