Fig 1: Effects of Wnt2 over-expression on astrocyte dedifferentiation and cortical neurogenesis.a Western-blotting of Wnt2 in ischemic cortex infected by pLenti-Luci (control) and Wnt2. b Western-blotting of Nestin and double-immunostaining of Nestin/Sox2 in ischemic cortex infected by pLenti-Luci (control) and Wnt2. Bar = 50 µm. N = 4 mice per group. c Representative images of Nestin/GFAP double-immunostaining in the bilateral cortices at 14 dpi with two ischemic injures infected by pLenti-Luci (left) and Wnt2 (right) respectively. Bar = 200 µm in upper panel. Bar = 50 µm in enlarged view. N = 4 mice per group. d Double-immunostaining and quantification of DCX/BrdU in ischemic cortex infected by pLenti-Luci and Wnt2. N = 3 mice per group. **P = 0.0075. Two-tailed Student’s t test. Bar = 50 µm. e Double-immunostaining and quantification of NeuN/BrdU in ischemic cortices infected by lentivirus expressing luciferase (control) or Wnt2. S1 and S2 are two representative sections showing one NeuN/BrdU-positive cell in each section. Arrows point to double-stained cells. Bar = 30 µm, and 5 µm in insert. ***P < 0.001. Two-tailed Student’s t test. N = 4 mice per group. Mean ± standard error (SE). Asterisks with bars connecting two groups indicate difference between these two groups.
Fig 2: Requirement of caspase-3 for Wnt2 activation and neurogenesis in ischemic cortex.a Combination of TUNEL staining and Wnt2 immunostaining (left panels), and Western-blotting of Wnt2 in the ischemic cortex of WT and caspase-3-/- mice (right panels). Expression of Wnt2 in the injured WT cortex is significantly increased, but remains unchanged in caspase-3-/- cortex. Dashed lines indicate lesion border. Bar = 30 µm. **P = 0.0061 (left panel), **P = 0.0019 (right panel). One-way ANOVA followed by Bonferroni’s post hoc comparisons tests. N = 3 mice per group. b Representative images of Nestin/ß-gal double immunostaining (left panels), and Western-blotting of ß-gal in the ischemic cortex of Topgal mice with or without caspase-3 mutation at 5 dpi (right panels). The expression of ß-gal in the ischemic cortex of Topgal:caspase-3+/+ mice is greatly enhanced, whereas it shows no notable change in Topgal:caspase-3-/- mice. Bar = 30 µm. **P = 0.0096 (left panel), **P = 0.0015 (right panel). One-way ANOVA followed by Bonferroni’s post hoc comparisons tests. N = 3–4 mice per group. c Double staining of BrdU with Nestin, and Western-blotting of Nestin in the ischemic WT and caspase-3-/- cortex. Notice the lower level of Nestin in the ischemic caspase-3-/- cortex. **P = 0.001 (left panel), **P = 0.0012 (right panel). One-way ANOVA followed by Bonferroni’s post hoc comparisons tests. N = 3–4 mice per group. d Double staining and quantification of BrdU/DCX. The numbers of BrdU/DCX-positive cells is significantly decreased in the caspase-3-/- cortex. Bars = 30 µm. **P = 0.0072 (left panel), **P = 0.0021 (right panel). Two-tailed Student’s t test. N = 3–4 per group. Arrows in (b, d) point to double-positive cells and arrowheads in (d) point to magnified double-positive cells. Mean ± standard error (SE). Asterisks with bars connecting two groups indicate difference between these two groups.
Fig 3: Effects of Wnt2 knockdown on astrocyte dedifferentiation and cortical neurogenesis.a Representative images of Nestin/GFAP double-immunostaining in the bilateral cortice with two ischemic injures infected by lentivirus expressing control shRNA (right) and Wnt2 shRNA (left), respectively. Bars = 200 µm in upper panel. Bar = 100 µm in enlarged view. b Western-blotting of Nestin and Sox2 in Wnt2 shRNA treated mice at 7 dpi. Wnt2 shRNA dramatically suppressed the expression of Nestin and Sox2 without obviously affecting the expression of GFAP. N = 5 mice per group. c Double-immunostaining and quantification of DCX/BrdU in ischemic cortices treated by control shRNA or Wnt2 shRNA. Arrowheads point to double-positive cells. Arrows point to magnified cells. Bar = 20 µm. **P = 0.0087. Two-tailed Student’s t test. N = 4 mice per group. d–f Forelimb activity assay of mice treated with control shRNA or Wnt2 shRNA. From 2 weeks on, the sliding scores and the asymmetric index of forelimb activity in Wnt2 shRNA treated group were much higher than control group, indicating worse functional recovery. N = 5 mice per group. P < 0.0001 (2w, 3w, 4w and 5w). Two-way RM ANOVA. a–c, asterisks with bars connecting two groups indicate difference between these two groups. e, f, P values indicate comparison of experimental group with control group of same time point. Mean ± standard error (SE).
Fig 4: Schematic graph of the major finding.Following cerebral ischemia, apoptotic neurons up-regulate Wnt2 protein via IRES mediated protein translation. Released Wnt2 activates Wnt/ß-catenin signaling in reactive astrocyte and triggers astrocytic dedifferentiation, which facilitated cortical neurogenesis.
Fig 5: Up-regulation of Wnt2 by apoptotic neurons and its involvement in Wnt//ß-catenin signaling activation.a Double-immunostaining of NeuN/Wnt2 in normal cortex. Bar = 50 µm. b, c Western-blotting of Wnt2 at different time points in ischemic cortex. Notice that Wnt2 protein increases quickly after ischemia. *P = 0.026 (1 h), 0.028 (2 h), 0.024 (4 h), 0.048 (8 h), 0.043 (12 h). One-way ANOVA followed by Bonferroni’s post hoc comparisons tests. N = 4 mice per group. d Combination of TUNEL staining and Wnt2 immunostaining at 24 hpi. Wnt2high immunoreactivity overlapped well with TUNEL-staining (arrowheads). Dashed line indicates lesion border and asterisk indicates lesion area. Bar = 30 µm. e Representative image of TUNEL/Wnt2/NeuN-triple immunostaining in the lesion area. Bar = 10 µm. f, g Western-blotting of Wnt2 in conditioned medium of normal cultured (NNCM), apoptotic (ANCM), Wnt2 shRNA pretreated apoptotic (shRNA ANCM) wild type and caspase-3-/- neurons. Apoptotic neurons increase Wnt2 release into the culture medium, and this increase is compromised by caspase-3 ablation and Wnt2 shRNA. BSA was used as internal control. *PNNCM vs ANCM = 0.012, *PANCM vs ShRNA-ANCM = 0.014. One-way ANOVA followed by Bonferroni’s post hoc comparisons tests. N = 3 batches of cells. h Western-blotting of active ß-catenin in the cytoplasmic and nuclear protein of astrocytes treated by NNCM, ANCM, AICM or shRNA ANCM. ANCM greatly increases the level of ß-catenin in nuclear protein. LMNB1 was used as nuclear protein control. i Double-immunostaining of GFAP/ß-gal in Topgal mice pretreated with Wnt2 shRNA. Arrows point to representative double-positive cells. Bar = 50 µm. j Western-blotting of ß-gal and Axin2 in ischemic cortex of Topgal mice treated with Wnt2 shRNA or control. Left: *Pipsi vs ipsi+Wnt2-shRNA = 0.038, *Pipsi+Con-shRNA vs ipsi+Wnt2-shRNA = 0.041, Right: *Pipsi vs ipsi+Wnt2-shRNA = 0.013, *Pipsi+Con-shRNA vs ipsi+Wnt2-shRNA = 0.016. One-way ANOVA followed by Bonferroni’s post hoc comparisons tests. N = 3 mice per group. Notice the less induction of ß-gal and Axin2 in Wnt2 shRNA pretreated cortex. Mean ± standard error (SE). Asterisks indicate the comparison with control group. Asterisks with bars connecting two groups indicate difference between these two groups.
Supplier Page from CUSABIO Technology LLC for Mouse Protein Wnt-2(WNT2) ELISA Kit