recombinant wnt2 protein  (Cusabio)

Journal: Advanced Science

Article Title: Optogenetic Stimulation of mPFC Alleviates White Matter Injury‐Related Cognitive Decline after Chronic Ischemia through Adaptive Myelination

doi: 10.1002/advs.202202976

Figure Lengend Snippet: Activation of glutamatergic neurons in the mPFC upregulates Wnt2 expression in the corpus callosum. a) Heatmap of the results of mPFC tissue microarray analysis between the BCAS mCherry and BCAS ChR2 groups at 2 months after surgery. n = 3 per group. b) Volcano plot of the results of mPFC tissue microarray analysis between the BCAS mCherry and BCAS ChR2 groups at 2 months after surgery (fold change > 2; p < 0.05). The DEGs are listed. n = 3 per group. c) Real‐time PCR for validation of the identified DEGs among the sham mCherry, BCAS mCherry, and BCAS ChR2 groups at 2 months after surgery. n = 7–8 per group. d) Real‐time PCR analysis of Wnt2 expression in the mPFC in the sham mCherry, BCAS mCherry, and BCAS hM3D groups at 2 months after surgery. n = 7–8 per group. e) Real‐time PCR analysis of Wnt2 expression in different types of primary cortical neural cells. n = 3 per group. f) Representative immunostaining of NeuN and Wnt2 in the mPFC in the sham mCherry, BCAS mCherry, and BCAS ChR2 groups at 2 months after surgery. Scale bar: 50 µm. g) Representative immunostaining of NF‐H and Wnt2 in the corpus callosum in the sham mCherry, BCAS mCherry, and BCAS ChR2 groups at 2 months after surgery. Scale bar: 35 µm. Quantification of immunostaining of Wnt2 in the h) mPFC and i) corpus callosum in the sham mCherry, BCAS mCherry, and BCAS ChR2 groups at 2 months after surgery. The fluorescence intensity in each image was normalized to that of NeuN and NF‐H. The measured values were normalized to the mean value of the BCAS group. n = 3 per group. j) Representative immunostaining of NeuN and Wnt2 in the mPFC in the sham mCherry, BCAS mCherry, and BCAS hM3D groups at 2 months after surgery. Scale bar: 50 µm. k) Representative immunostaining of NF‐H and Wnt2 in the corpus callosum in the sham mCherry, BCAS mCherry, and BCAS hM3D groups at 2 months after surgery. Scale bar: 35 µm. Quantification of Wnt2 immunostaining in the l) mPFC and m) corpus callosum in the sham mCherry, BCAS mCherry, and BCAS hM3D groups at 2 months after surgery. The fluorescence intensity in each image was normalized to that of NeuN and NF‐H. The measured values were normalized to the mean value of the BCAS group. n = 3 per group. The data are presented as the mean ± SEM. p ‐values were determined by the Kruskal–Wallis test with Dunn's post‐hoc analysis in (d); by 1‐way ANOVA with Tukey's post‐hoc analysis in (c) and (e); and by Student's t ‐test in (h), (i), (l), and (m). * p < 0.05, ** p < 0.01.

Article Snippet: Afterward, 50 ng mL −1 T3 (Thermo Fisher Scientific, USA), 200 ng mL −1 recombinant Wnt2 protein (Cusabio, China), 100 ng mL −1 recombinant Dkk1 protein (Cusabio, China) and basic medium DMEM F12 supplemented with 2% B27, 100 U mL −1 antibiotics, and 20 ng mL −1 CNTF (Thermo Fisher Scientific, USA) were applied to induce differentiation.

Techniques: Activation Assay, Expressing, Microarray, Real-time Polymerase Chain Reaction, Biomarker Discovery, Immunostaining, Fluorescence

Journal: Advanced Science

Article Title: Optogenetic Stimulation of mPFC Alleviates White Matter Injury‐Related Cognitive Decline after Chronic Ischemia through Adaptive Myelination

doi: 10.1002/advs.202202976

Figure Lengend Snippet: Overexpression of Wnt2 in mPFC glutamatergic neurons alleviates myelin injury and improves cognition. Results of the a) Y‐maze and b) T‐maze tests showing the spontaneous alternation percentage in the control and Camk2a‐Wnt2 groups at 2 months after surgery. n = 15–16 per group. Results of the open field test showing c) the total distance travelled and d) time spent in the corner area and e) center area in the control and Camk2a‐Wnt2 groups at 2 months after surgery. n = 15–16 per group. f) Heatmaps generated from DTI axial views of FA acquired from the control and Camk2a‐Wnt2 groups at 2 months after surgery. g) Quantification of FA values in the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. The measured values were normalized to the mean value of the control group. n = 11 per group. h) Representative images of Black‐gold staining and immunostaining of MBP and MAG in the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. Scale bar: 200 µm. Quantification of i) Black‐gold staining and immunostaining of j) MBP and k) MAG in the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. n = 4 per group. l) Representative TEM images of the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. Scale bar: 1 µm. m) The percentage of myelinated axons in the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. n = 4 per group. n) Scatterplots of the myelin g‐ratio as a function of the axon diameter in the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. Axons were selected from 4 mice per group for measurement. n = 138 and n = 121 measured axons for each group, respectively. o) Representative immunostaining of CC1 and Olig2 in the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. Scale bar: 50 µm. Density of p) CC1 + Olig2 + cells and q) CC1 − Olig2 + cells in the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. n = 5 per group. r) Proportion of CC1 + /Olig2 + cells in the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. n = 5 per group. s) Immunoblot bands and t) quantification of MBP and u) MAG expression in OPCs in the control, T3, and T3+ rmWnt2 groups. The intensity of each immunoblot band was normalized to that of the β ‐actin band. The measured values were normalized to the mean value of the control group. n = 3 per group. The data are presented as the mean ± SEM. p ‐values were determined by Student's t ‐test in (b–e), (i–k), (m), (p–r), and (t); by the Mann–Whitney test in (a), (g), and (n); and by 1‐way ANOVA with Tukey's post‐hoc analysis in (u). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Afterward, 50 ng mL −1 T3 (Thermo Fisher Scientific, USA), 200 ng mL −1 recombinant Wnt2 protein (Cusabio, China), 100 ng mL −1 recombinant Dkk1 protein (Cusabio, China) and basic medium DMEM F12 supplemented with 2% B27, 100 U mL −1 antibiotics, and 20 ng mL −1 CNTF (Thermo Fisher Scientific, USA) were applied to induce differentiation.

Techniques: Over Expression, Control, Generated, Staining, Immunostaining, Western Blot, Expressing, MANN-WHITNEY

Journal: Cell reports

Article Title: In vitro atlas of dorsal spinal interneurons reveals Wnt signaling as a critical regulator of progenitor expansion

doi: 10.1016/j.celrep.2022.111119

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human recombinant Wnt2 , Millipore , SRP-6560.

Techniques: Recombinant, Membrane, Saline, Software, Gene Expression, Variant Assay

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Serum Wnt2 and Wnt4 are increased in patients with AMI and correlated to the increased risk of adverse outcomes of patients (a, b) ELISA analysis of serum Wnt2 and Wnt4 level in a total of 109 patients with AMI and 56 non-AMI patients. Data are expressed as means±SD, * p < 0.05, ** p < 0.01, *** p < 0.001 by Student's t test. (c, d) Kaplan–Meier incidence of MACEs in one year according to high or low level of serum Wnt2 or Wnt4. Wnt2 and Wnt4 were dichotomized into 2 categories with categorical analysis including higher than median and lower than median. Wnt2 high: ≥0.86 (ng/mL); Wnt2 low: < 0.86(ng/mL); Wnt4 high:≥86.2(pg/mL); Wnt4 low: < 86.2(pg/mL). MACEs: major adverse cardiovascular events. Estimated HR, 95% CIs, and p values were calculated. Statistics: The cumulative incidence of the MACE was determined by the Kaplan–Meier method, and the difference between groups was compared using the log-rank test. MACEs: major adverse cardiovascular events. Estimated HR, 95% CIs, and p values were calculated.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Univariate and multivariable adjusted Cox proportional hazard regression models of incident MACEs with serum Wnt2 and Wnt4 in AMI patients.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques:

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Serum and cardiac Wnt2 and Wnt4 levels are increased in mice following MI. (a) Western blot analysis of the expression of Wnt2, Wnt4, Col1, Col3 and TGF- β1 in the border zone of infarcted area at different time-points (3d, 7d, 14d, 28d) following MI. Sham operation was used as control. Values were expressed as means±S.E.M; * p < 0.05, ** p < 0.01, *** p < 0.001 vs sham group, n = 5/group. (b) Western blot analysis of serum Wnt2 and Wnt4 from sham group and different time-points (3d,7d,14d,28d) after MI. Values were expressed as means±S.E.M; * p < 0.05, ** p < 0.01, *** p < 0.001 vs sham group, n = 4/group. (c) Wnt2 and Wnt4 expression were analyzed by Western blot analysis in cultured neonatal rat cardiac fibroblasts (NRCFs) at different time-points (1 h,3 h,6 h,12 h,24 h) in response to hypoxia. Normaxia group was used as control. Values were expressed as means±S.E.M; * p < 0.05, ** p < 0.01, *** p < 0.001 vs control group. n = 4/group. (d) The representative images of Wnt2 and Wnt4 expression with Western blot analysis in conditional medium from neonatal rat cardiac fibroblasts (NRCFs) of control group and at different time-points (1 h,3 h,6 h,12 h,24 h) after hypoxia. In (b) and (d), a conventional Coomassie staining of polyvinylidene fluoride (PVDF) membranes showed the total protein load which was as the internal control (Loading control). The experiment was repeated for three times. MI: Myocardial infarction; CON: control. Statistics: One-way ANOVA with post-hoc Tukey test.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Western Blot, Expressing, Control, Cell Culture, Staining

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Knockdown of Wnt2/Wnt4 suppresses cardiac dysfunction and fibrosis post-MI. 8-10 weeks old male mice were injected with shWnt2/4-AAV9 (shWnt2/4) or shScramble-AAV9 (shNC, as control) by tail vein at 3 weeks before sham or MI operation. (a) Wnt2 and Wnt4 levels were determined in the non-infarcted area by western blot method on day 28 post-MI. Values were expressed as means±S.E.M; *** p < 0.001 vs shNC group; n = 5/each group. Statistics: Two-way ANOVA with a Bonferroni post hoc test. (b) Echocardiographic analysis of mice at day 0, day7, day14 and day28 after MI. Left lane: Representative images of M-mode echocardiogram captured on the 28th day post-MI. Right lane: quantitative analysis of LVEF, FS; LVEF: Left ventricular ejection fraction; FS, fraction shortening. Values were presented as means±S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001 vs shNC group; n = 6/group. Statistics: Student's t test. (c) Hemodynamics analysis of dp/dt, -dp/dt, LVEDP and Tau at 21 days after MI in mice. Values were expressed as means±S.E.M. * p < 0.05;** p < 0.01;*** p < 0.001, n = 4-8 in each group. Statistics: Two-way ANOVA with a Bonferroni post hoc test. (d) Cardiac fibrosis was examined by Masson staining at 28 days after MI in mice. Bar =1000 μm. Values were expressed as means±S.E.M. ** p < 0.01 vs shNC group, n = 6 in each group. Statistics: Student's t test. (e) Col1, Col3, MMP2, MMP9 and α-SMA protein levels were analyzed by Western blot in border zone of infarcted area on day 28 post-MI. Values were presented as means±S.E.M .* p < 0.05, ** p < 0.01,*** p < 0.001; n = 4 in each group. Statistics: Two-way ANOVA with a Bonferroni post hoc test.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Knockdown, Injection, Control, Western Blot, Staining

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Wnt2 or Wnt4 is involved in fibroblasts activation in response to hypoxia. (a,b) Western blot analysis of the expressions of Wnt2, Wnt4, Col1, Col3, TGFβ1, MMP9 MMP2, p-smad2/3, CTGF and α-SMA protein in neonatal rat cardiac fibroblasts (NRCFs). These NRCFs were pretreated with siRNA targeted Wnt2 or Wnt4 (si-Wnt2 or si-Wnt4) or si-Scramble (si-Scram) for 24 h, and then exposed to hypoxia or normoxia (Control) for 3 h. p: pro-TGF-β1; m: mature or active TGF-β1; TGF-β1 mature form was analyzed. Values were described as means±S.E.M; * p < 0.05, ** p < 0.01, *** p < 0.001; n = 3/group. Statistics: Two-way ANOVA with a Bonferroni post hoc test. c: Scratch assay for migratory ability of cardiac fibroblasts pre-transfected with si-scram (CON), si-Wnt2 or si-Wnt4 followed by hypoxia. The representative images were showed at 0 h and 12 h after hypoxia. The bar=20 um; Values were quantitified and expressed as means±S.E.M. ** p < 0.01, *** p < 0.001 vs si-scram group; n = 4/group. Statistics: One-way ANOVA with post-hoc Tukey test.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Activation Assay, Western Blot, Control, Wound Healing Assay, Transfection

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Wnt2 or Wnt4 activates NF-κB pathway signaling and enhances the expression of Fzd4/Fzd2 in neonatal rat cardiac fibroblasts (NRCFs) in response to hypoxia. (a) The expressions of p-p65, P65, Fzd2 and Fzd4 in NRCFs were detected by Western blot analysis. n = 4/group. (b) The expressions of p65 and β-catenin were detected in nucleus of NRCFs by western blot. n = 3/group. (a,b) These NRCFs were pretreated with siRNA targeted Wnt2 or Wnt4 (si-Wnt2 or si-Wnt4) or si-Scramble (si-Scram) for 24 h, and then exposed to hypoxia or normoxia (Control) for 3 h. Values were expressed as means±S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001. (c) The expressions of p-p65, p65, Fzd 2 and Fzd 4 were detected in non-infarcted area by Western blot analysis. 8-10 weeks old male mice were intravenously injected with shWnt2/4-AAV9 (shWnt2/4) or shScramble-AAV9 (shNC) at 3 weeks before sham or MI operation. These proteins were detected at day 28 post MI or sham operation. n = 4 in each group. Values were expressed as means±S.E.M; ** p < 0.01, *** p < 0.001. Statistics: Two-way ANOVA with a Bonferroni post hoc test.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Expressing, Western Blot, Control, Injection

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: NF-κB inhibitor ameliorates Wnt2/Wnt4-induced pro-fibrotic effects in neonatal rat cardiac fibroblasts (NRCFs). (a,b) The expressions of Col1, Col3, active β-catenin, MMP2, MMP9, p65, p-p65 and TGFβ1 were measured in NRCFs by Western blot analysis. These NRCFs were pretreated with JSH-23(NF-κB inhibitor, 10μM) or the same volume of DMSO for 1 h, followed by PBS, human recombinant Wnt2 (20 ng/ml) or Wnt4 (50 ng/ml) treatment for 24 h. Values were expressed as means±S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001; n = 3 in each group. Statistics: Two-way ANOVA with a Bonferroni post hoc test.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Western Blot, Recombinant

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Wnt/β-catenin inhibitor relieves Wnt2/Wnt4-induced pro-fibrotic effects in neonatal rat cardiac fibroblasts (NRCFs). (a,b) The expressions of Col1, Col3, active β-catenin, p65, TGFβ1, Fzd2, Fzd4 and p-p65 were measured in NRCFs by Western blot analysis. These NRCFs were pretreated with ICG-001(β-catenin inhibitor, 10 μM) or the same volume of DMSO for 1 h, followed by PBS, human recombinant Wnt2 (20 ng/ml) or Wnt4 (50 ng/ml) treatment for 24 h. Values were expressed as means±S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001, n = 3 in each group. Statistics: Two-way ANOVA with a Bonferroni post hoc test.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Western Blot, Recombinant

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Knockdown of Fzd2 or Fzd4 attenuates Wnt4- or Wnt2-induced pro-fibrotic effects in neonatal rat cardiac fibroblasts (NRCFs). (a,b) Western blot analysis of the expressions of Fzd2, Col1, Col3, active β-catenin, MMP2, MMP9, TGFβ1, and p-65 in NRCFs. Values were expressed as means±S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001; n = 3 in each group. Statistics: Two-way ANOVA with a Bonferroni post hoc test. (a) NRCFs were pretreated with siRNA targeted Fzd2 (si- Fzd2) or si-Scramble (si-Scram) for 24 h, followed by PBS, human recombinant Wnt4 (50 ng/ml) treatment for another 24 h. (b) NRCFs were pretreated with siRNA targeted Fzd4 (si- Fzd4) or si-Scramble (si-Scram) for 24 h, followed by PBS, human recombinant Wnt2 (20 ng/ml) treatment for another 24 h.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Knockdown, Western Blot, Recombinant

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Knockdown of LRP6 attenuates Wnt2- or Wnt4-induced pro-fibrotic effects in neonatal rat cardiac fibroblasts (NRCFs). (a,b) Western blot analysis of the expressions of Col1, Col3, active β-catenin, TGFβ1, LRP6 and p-65 in NRCFs. Cultured NRCFs were transfected with lenti-shScr (shScr) or lenti-LRP6(shLRP6) for 48 h and then followed by PBS, human recombinant Wnt2 (20 ng/ml) or Wnt4(50 ng/ml) treatment for another 24 h. Values were expressed as means±S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001. n = 3 /group. Statistics: Two-way ANOVA with a Bonferroni post hoc test.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Knockdown, Western Blot, Cell Culture, Transfection, Recombinant

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: A proposed model of showing how Wnt2 and Wnt4 promote cardiac fibrosis by activation of β-catenin/NF-κB trough LRP6 and Fzds signaling following MI. MI induced the increased expression or secretion of Wnt2 and Wnt4 in cardiaomyocytes (CMs) or cardiac fibroblasts (CFs), elevated Wnt2 and Wnt4 promote fibrotic effects by activation of β-catenin/NF-κB signaling dependently on the cooperation of Fzd4 or Fzd2 and LRP6 signaling in cardiac fibroblasts, which contributes to cardiac fibrosis and dysfunction post-MI. Thus high Wnt2 and Wnt4 are independently associated with adverse outcome in AMI patients.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Activation Assay, Expressing

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Serum Wnt2 and Wnt4 are increased in patients with AMI and correlated to the increased risk of adverse outcomes of patients (a, b) ELISA analysis of serum Wnt2 and Wnt4 level in a total of 109 patients with AMI and 56 non-AMI patients. Data are expressed as means±SD, * p < 0.05, ** p < 0.01, *** p < 0.001 by Student's t test. (c, d) Kaplan–Meier incidence of MACEs in one year according to high or low level of serum Wnt2 or Wnt4. Wnt2 and Wnt4 were dichotomized into 2 categories with categorical analysis including higher than median and lower than median. Wnt2 high: ≥0.86 (ng/mL); Wnt2 low: < 0.86(ng/mL); Wnt4 high:≥86.2(pg/mL); Wnt4 low: < 86.2(pg/mL). MACEs: major adverse cardiovascular events. Estimated HR, 95% CIs, and p values were calculated. Statistics: The cumulative incidence of the MACE was determined by the Kaplan–Meier method, and the difference between groups was compared using the log-rank test. MACEs: major adverse cardiovascular events. Estimated HR, 95% CIs, and p values were calculated.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Univariate and multivariable adjusted Cox proportional hazard regression models of incident MACEs with serum Wnt2 and Wnt4 in AMI patients.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques:

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Serum and cardiac Wnt2 and Wnt4 levels are increased in mice following MI. (a) Western blot analysis of the expression of Wnt2, Wnt4, Col1, Col3 and TGF- β1 in the border zone of infarcted area at different time-points (3d, 7d, 14d, 28d) following MI. Sham operation was used as control. Values were expressed as means±S.E.M; * p < 0.05, ** p < 0.01, *** p < 0.001 vs sham group, n = 5/group. (b) Western blot analysis of serum Wnt2 and Wnt4 from sham group and different time-points (3d,7d,14d,28d) after MI. Values were expressed as means±S.E.M; * p < 0.05, ** p < 0.01, *** p < 0.001 vs sham group, n = 4/group. (c) Wnt2 and Wnt4 expression were analyzed by Western blot analysis in cultured neonatal rat cardiac fibroblasts (NRCFs) at different time-points (1 h,3 h,6 h,12 h,24 h) in response to hypoxia. Normaxia group was used as control. Values were expressed as means±S.E.M; * p < 0.05, ** p < 0.01, *** p < 0.001 vs control group. n = 4/group. (d) The representative images of Wnt2 and Wnt4 expression with Western blot analysis in conditional medium from neonatal rat cardiac fibroblasts (NRCFs) of control group and at different time-points (1 h,3 h,6 h,12 h,24 h) after hypoxia. In (b) and (d), a conventional Coomassie staining of polyvinylidene fluoride (PVDF) membranes showed the total protein load which was as the internal control (Loading control). The experiment was repeated for three times. MI: Myocardial infarction; CON: control. Statistics: One-way ANOVA with post-hoc Tukey test.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Western Blot, Expressing, Control, Cell Culture, Staining

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Knockdown of Wnt2/Wnt4 suppresses cardiac dysfunction and fibrosis post-MI. 8-10 weeks old male mice were injected with shWnt2/4-AAV9 (shWnt2/4) or shScramble-AAV9 (shNC, as control) by tail vein at 3 weeks before sham or MI operation. (a) Wnt2 and Wnt4 levels were determined in the non-infarcted area by western blot method on day 28 post-MI. Values were expressed as means±S.E.M; *** p < 0.001 vs shNC group; n = 5/each group. Statistics: Two-way ANOVA with a Bonferroni post hoc test. (b) Echocardiographic analysis of mice at day 0, day7, day14 and day28 after MI. Left lane: Representative images of M-mode echocardiogram captured on the 28th day post-MI. Right lane: quantitative analysis of LVEF, FS; LVEF: Left ventricular ejection fraction; FS, fraction shortening. Values were presented as means±S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001 vs shNC group; n = 6/group. Statistics: Student's t test. (c) Hemodynamics analysis of dp/dt, -dp/dt, LVEDP and Tau at 21 days after MI in mice. Values were expressed as means±S.E.M. * p < 0.05;** p < 0.01;*** p < 0.001, n = 4-8 in each group. Statistics: Two-way ANOVA with a Bonferroni post hoc test. (d) Cardiac fibrosis was examined by Masson staining at 28 days after MI in mice. Bar =1000 μm. Values were expressed as means±S.E.M. ** p < 0.01 vs shNC group, n = 6 in each group. Statistics: Student's t test. (e) Col1, Col3, MMP2, MMP9 and α-SMA protein levels were analyzed by Western blot in border zone of infarcted area on day 28 post-MI. Values were presented as means±S.E.M .* p < 0.05, ** p < 0.01,*** p < 0.001; n = 4 in each group. Statistics: Two-way ANOVA with a Bonferroni post hoc test.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Knockdown, Injection, Control, Western Blot, Staining

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Wnt2 or Wnt4 is involved in fibroblasts activation in response to hypoxia. (a,b) Western blot analysis of the expressions of Wnt2, Wnt4, Col1, Col3, TGFβ1, MMP9 MMP2, p-smad2/3, CTGF and α-SMA protein in neonatal rat cardiac fibroblasts (NRCFs). These NRCFs were pretreated with siRNA targeted Wnt2 or Wnt4 (si-Wnt2 or si-Wnt4) or si-Scramble (si-Scram) for 24 h, and then exposed to hypoxia or normoxia (Control) for 3 h. p: pro-TGF-β1; m: mature or active TGF-β1; TGF-β1 mature form was analyzed. Values were described as means±S.E.M; * p < 0.05, ** p < 0.01, *** p < 0.001; n = 3/group. Statistics: Two-way ANOVA with a Bonferroni post hoc test. c: Scratch assay for migratory ability of cardiac fibroblasts pre-transfected with si-scram (CON), si-Wnt2 or si-Wnt4 followed by hypoxia. The representative images were showed at 0 h and 12 h after hypoxia. The bar=20 um; Values were quantitified and expressed as means±S.E.M. ** p < 0.01, *** p < 0.001 vs si-scram group; n = 4/group. Statistics: One-way ANOVA with post-hoc Tukey test.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Activation Assay, Western Blot, Control, Wound Healing Assay, Transfection

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Wnt2 or Wnt4 activates NF-κB pathway signaling and enhances the expression of Fzd4/Fzd2 in neonatal rat cardiac fibroblasts (NRCFs) in response to hypoxia. (a) The expressions of p-p65, P65, Fzd2 and Fzd4 in NRCFs were detected by Western blot analysis. n = 4/group. (b) The expressions of p65 and β-catenin were detected in nucleus of NRCFs by western blot. n = 3/group. (a,b) These NRCFs were pretreated with siRNA targeted Wnt2 or Wnt4 (si-Wnt2 or si-Wnt4) or si-Scramble (si-Scram) for 24 h, and then exposed to hypoxia or normoxia (Control) for 3 h. Values were expressed as means±S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001. (c) The expressions of p-p65, p65, Fzd 2 and Fzd 4 were detected in non-infarcted area by Western blot analysis. 8-10 weeks old male mice were intravenously injected with shWnt2/4-AAV9 (shWnt2/4) or shScramble-AAV9 (shNC) at 3 weeks before sham or MI operation. These proteins were detected at day 28 post MI or sham operation. n = 4 in each group. Values were expressed as means±S.E.M; ** p < 0.01, *** p < 0.001. Statistics: Two-way ANOVA with a Bonferroni post hoc test.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Expressing, Western Blot, Control, Injection

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: NF-κB inhibitor ameliorates Wnt2/Wnt4-induced pro-fibrotic effects in neonatal rat cardiac fibroblasts (NRCFs). (a,b) The expressions of Col1, Col3, active β-catenin, MMP2, MMP9, p65, p-p65 and TGFβ1 were measured in NRCFs by Western blot analysis. These NRCFs were pretreated with JSH-23(NF-κB inhibitor, 10μM) or the same volume of DMSO for 1 h, followed by PBS, human recombinant Wnt2 (20 ng/ml) or Wnt4 (50 ng/ml) treatment for 24 h. Values were expressed as means±S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001; n = 3 in each group. Statistics: Two-way ANOVA with a Bonferroni post hoc test.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Western Blot, Recombinant

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Wnt/β-catenin inhibitor relieves Wnt2/Wnt4-induced pro-fibrotic effects in neonatal rat cardiac fibroblasts (NRCFs). (a,b) The expressions of Col1, Col3, active β-catenin, p65, TGFβ1, Fzd2, Fzd4 and p-p65 were measured in NRCFs by Western blot analysis. These NRCFs were pretreated with ICG-001(β-catenin inhibitor, 10 μM) or the same volume of DMSO for 1 h, followed by PBS, human recombinant Wnt2 (20 ng/ml) or Wnt4 (50 ng/ml) treatment for 24 h. Values were expressed as means±S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001, n = 3 in each group. Statistics: Two-way ANOVA with a Bonferroni post hoc test.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Western Blot, Recombinant

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Knockdown of Fzd2 or Fzd4 attenuates Wnt4- or Wnt2-induced pro-fibrotic effects in neonatal rat cardiac fibroblasts (NRCFs). (a,b) Western blot analysis of the expressions of Fzd2, Col1, Col3, active β-catenin, MMP2, MMP9, TGFβ1, and p-65 in NRCFs. Values were expressed as means±S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001; n = 3 in each group. Statistics: Two-way ANOVA with a Bonferroni post hoc test. (a) NRCFs were pretreated with siRNA targeted Fzd2 (si- Fzd2) or si-Scramble (si-Scram) for 24 h, followed by PBS, human recombinant Wnt4 (50 ng/ml) treatment for another 24 h. (b) NRCFs were pretreated with siRNA targeted Fzd4 (si- Fzd4) or si-Scramble (si-Scram) for 24 h, followed by PBS, human recombinant Wnt2 (20 ng/ml) treatment for another 24 h.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Knockdown, Western Blot, Recombinant

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: Knockdown of LRP6 attenuates Wnt2- or Wnt4-induced pro-fibrotic effects in neonatal rat cardiac fibroblasts (NRCFs). (a,b) Western blot analysis of the expressions of Col1, Col3, active β-catenin, TGFβ1, LRP6 and p-65 in NRCFs. Cultured NRCFs were transfected with lenti-shScr (shScr) or lenti-LRP6(shLRP6) for 48 h and then followed by PBS, human recombinant Wnt2 (20 ng/ml) or Wnt4(50 ng/ml) treatment for another 24 h. Values were expressed as means±S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001. n = 3 /group. Statistics: Two-way ANOVA with a Bonferroni post hoc test.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Knockdown, Western Blot, Cell Culture, Transfection, Recombinant

Journal: EBioMedicine

Article Title: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction

doi: 10.1016/j.ebiom.2021.103745

Figure Lengend Snippet: A proposed model of showing how Wnt2 and Wnt4 promote cardiac fibrosis by activation of β-catenin/NF-κB trough LRP6 and Fzds signaling following MI. MI induced the increased expression or secretion of Wnt2 and Wnt4 in cardiaomyocytes (CMs) or cardiac fibroblasts (CFs), elevated Wnt2 and Wnt4 promote fibrotic effects by activation of β-catenin/NF-κB signaling dependently on the cooperation of Fzd4 or Fzd2 and LRP6 signaling in cardiac fibroblasts, which contributes to cardiac fibrosis and dysfunction post-MI. Thus high Wnt2 and Wnt4 are independently associated with adverse outcome in AMI patients.

Article Snippet: Human Wnt2 and Wnt4 ELISA kits were purchased from CUSABIO company (Cat#: CSB-EL026133HU, CSB-EL026137HU, Wuhan, China) and used following the manufacturer's instructions.

Techniques: Activation Assay, Expressing