Journal: Molecular Cancer Research

Article Title: Demethylation of the SFRP4 Promoter Drives Gastric Cancer Progression via the Wnt Pathway

doi: 10.1158/1541-7786.mcr-20-0933

Figure Lengend Snippet: Figure 5. SFRP4 plays an antagonistic role on biology function of SFRP1. A, The expression of Wnt ligands (Wnt2, Wnt3a, Wnt4, Wnt5A) in different gas- tric cancer cells was examined by Western blotting. B, The binding activ- ity of Wnt5A to its receptor was exam- ined by flow cytometry in the absence or presence of recombinant SFRP4. C, Co-IP experiments in MGC80–3cells were performed using anti-SFRP4 or anti-b-catenin antibodies in the absence or presence of recombinant Wnt5a. IgG was used as a negative control. D, The Wnt pathway markers (GSK-3b, b-catenin, c-Jun, LEF1, CD44, and MMP7) were examined in MGC80–3 cell after treating with recombinant SFRP1 (100 ng/mL), recombinant SFRP4 (100 ng/mL), or both for 48 hours. E, Afterward, com- bination of recombinant SFRP1 and recombinant SFRP4 were addedto the MGC80–3 cell in a concentration gra- dient (1:0, 1:0.5, 1:1, 1:2, 1:3) for 48 hours before harvesting for Western blotting analysis. F, The effects of recombinant SFRP1 and SFRP4 on the proliferation of MGC80–3 and AGS cellswere exam- ined by CCK-8 assay. , P < 0.001. r, recombinant; NC, negative control. In (A–E), the images were representa- tive of three independent experi- ments. In F, the experiments were repeated three times. Statistical sig- nificance was determined by one-way ANOVA test (F).

Article Snippet: The recombinant human SFRP1 (1384-SF), SFRP4 (1827-SF) and Wnt5a(645-WN) protein were purchased from R&D Systems.

Techniques: Expressing, Western Blot, Binding Assay, Cytometry, Recombinant, Co-Immunoprecipitation Assay, Negative Control, Concentration Assay, CCK-8 Assay

Journal:

Article Title: VASCULAR INJURY DURING ELEVATED GLUCOSE CAN BE MITIGATED BY ERYTHROPOIETIN AND WNT SIGNALING

doi:

Figure Lengend Snippet: (A)Recombinant human Wnt1 protein (100 ng/ml) was applied to EC cultures 1 hour prior to the exposure of D-glucose (25 mM) (HG) and cell survival was determined 48 hours later. Representative images illustrate increased trypan blue staining during elevated glucose, but administration of Wnt1 significantly decreased trypan blue uptake by ECs. In contrast, application of Wnt1 antibody (Wnt1Ab, 1 μg/ml) 30 min prior to the administration of Wnt1 protein antagonized the ability of Wnt1 to significantly reduce trypan blue uptake in ECs during elevated glucose exposure. (B) Wnt1 (100 ng/ml) administration significantly increased EC survival when compared with cultures exposed to elevated glucose alone (*p<0.01 vs. HG). In contrast, application of Wnt1 antibody (Wnt1Ab, 1 μg/ml) 30 min prior to the administration of Wnt1 protein blocked Wnt1 cytoprotection in ECs during elevated glucose (†p<0.01 vs. Wnt1/HG). (C) Recombinant human Wnt1 protein (100 ng/ml) was administered to EC cultures 1 hour prior to the exposure of D-glucose (25 mM) (HG) and nuclear DNA fragmentation with TUNEL was determined 48 hours later. Representative images illustrate increased TUNEL staining during elevated glucose, but administration of Wnt1 significantly decreased TUNEL labeling in ECs. In contrast, application of Wnt1 antibody (Wnt1Ab, 1 μg/ml) 30 min prior to the administration of Wnt1 protein antagonized the ability of Wnt1 to significantly reduce TUNEL labeling in ECs during elevated glucose exposure. (D) Wnt1 (100 ng/ml) administration significantly decreased EC nuclear DNA degradation when compared with cultures exposed to elevated glucose alone (*p<0.01 vs. HG). In contrast, application of Wnt1 antibody (Wnt1Ab, 1 μg/ml) 30 min prior to the administration of Wnt1 protein prevented Wnt1 from reducing nuclear DNA degradation in ECs during elevated glucose (†p<0.01 vs. Wnt1/HG). In all cases, each data point represents the mean and SEM and control = untreated EC cultures.

Article Snippet: For treatments applied 1 hour prior to elevated glucose concentrations, application of erythropoietin (EPO) (R&D Systems, Minneapolis, MN), human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN), a mouse monoclonal anti body against Wnt1 (R&D Systems, 0olis, MN), or the glycogen synthase kinase (GSK)-3β inhibitor SB216763 [3-(2,4-Dichlorophenyl)-4-(1-methyl- 1 H-indol-3-yl)- 1 H-pyrrole-2,5-dione] (SB21) (Tocris, Ellisville, MO) were continuous.

Techniques: Recombinant, Staining, TUNEL Assay, Labeling, Control

Journal:

Article Title: VASCULAR INJURY DURING ELEVATED GLUCOSE CAN BE MITIGATED BY ERYTHROPOIETIN AND WNT SIGNALING

doi:

Figure Lengend Snippet: (A)EPO (10 ng/ml) was applied to EC cultures 1 hour prior to the exposure of D-glucose (25 mM) (HG) and cell survival was determined 48 hours later. Representative images illustrate increased trypan blue staining during elevated glucose, but administration of EPO significantly decreased trypan blue uptake by ECs. In contrast, application of Wnt1 antibody (Wnt1Ab, 1 μg/ml) 30 min prior to the administration of EPO antagonized the ability of EPO to prevent trypan blue uptake in ECs during elevated glucose exposure. (B) EPO (10 ng/ml) application significantly increased EC survival when compared with cultures exposed to elevated glucose alone (*p<0.01 vs. HG). In contrast, application of Wnt1 antibody (Wnt1Ab, 1 μg/ml) 30 min prior to the EPO treatment blocked Wnt1 cytoprotection in ECs during elevated glucose (†p<0.01 vs. EPO/HG). (C) EPO (10 ng/ml) was applied to EC cultures 1 hour prior to the exposure of D-glucose (25 mM) (HG) and nuclear DNA fragmentation with TUNEL was determined 48 hours later. Representative images illustrate increased TUNEL staining during elevated glucose, but administration of EPO significantly decreased nuclear DNA fragmentation as demonstrated by reduced TUNEL labeling in ECs. In contrast, application of Wnt1 antibody (Wnt1Ab, 1 μg/ml) 30 min prior to the administration of EPO prevented EPO from significantly reducing TUNEL labeling in ECs during elevated glucose exposure. (D) EPO (10 ng/ml) administration significantly decreased EC nuclear DNA degradation when compared with cultures exposed to elevated glucose alone (*p<0.01 vs. HG). In contrast, application of Wnt1 antibody (Wnt1Ab, 1 μg/ml) 30 min prior to EPO application prevented EPO from reducing nuclear DNA degradation in ECs during elevated glucose (†p<0.01 vs. EPO/HG). In all cases, each data point represents the mean and SEM and control = untreated EC cultures.

Article Snippet: For treatments applied 1 hour prior to elevated glucose concentrations, application of erythropoietin (EPO) (R&D Systems, Minneapolis, MN), human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN), a mouse monoclonal anti body against Wnt1 (R&D Systems, 0olis, MN), or the glycogen synthase kinase (GSK)-3β inhibitor SB216763 [3-(2,4-Dichlorophenyl)-4-(1-methyl- 1 H-indol-3-yl)- 1 H-pyrrole-2,5-dione] (SB21) (Tocris, Ellisville, MO) were continuous.

Techniques: Staining, TUNEL Assay, Labeling, Control

Journal:

Article Title: VASCULAR INJURY DURING ELEVATED GLUCOSE CAN BE MITIGATED BY ERYTHROPOIETIN AND WNT SIGNALING

doi:

Figure Lengend Snippet: EC protein extracts (50 μg/lane) were immunoblotted with anti-Wnt1 (A and B), anti-phosphorylated glycogen synthase kinase-3β (anti-p-GSK-3β) (C and D). Representative images of Western blot detection for Wnt1 (A) and p-GSK-3β (C) were performed at 6, 24, and 48 hour time intervals following administration of elevated D-glucose (25 mM) (HG). Wnt1 and p-GSK-3β expression increased at 6 and 24 hours following exposure to high glucose, but expression of these proteins was lost 48 hours following elevated glucose (*p< 0.01 vs. 6 hours or 24 hours HG). Application of EPO (10 ng/ml) 1 hour prior to the administration of elevated glucose significantly increased Wnt1 (B) (*p< 0.01 vs. HG) and p-GSK-3β (D) (*p< 0.01 vs. HG) expression 48 hours following elevated glucose treatment. In all cases, each data point represents the mean and SEM and control = untreated EC cultures.

Article Snippet: For treatments applied 1 hour prior to elevated glucose concentrations, application of erythropoietin (EPO) (R&D Systems, Minneapolis, MN), human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN), a mouse monoclonal anti body against Wnt1 (R&D Systems, 0olis, MN), or the glycogen synthase kinase (GSK)-3β inhibitor SB216763 [3-(2,4-Dichlorophenyl)-4-(1-methyl- 1 H-indol-3-yl)- 1 H-pyrrole-2,5-dione] (SB21) (Tocris, Ellisville, MO) were continuous.

Techniques: Western Blot, Expressing, Control

Journal:

Article Title: VASCULAR INJURY DURING ELEVATED GLUCOSE CAN BE MITIGATED BY ERYTHROPOIETIN AND WNT SIGNALING

doi:

Figure Lengend Snippet: Primary ECs were exposed to elevated D-glucose (25 mM) (HG) and EC survival or nuclear DNA fragmentation were determined 48 hours following elevated glucose exposure. (A and C) Elevated glucose resulted in a significant decrease EC survival and a significant increase in nuclear DNA fragmentation in ECs. Application of the GSK-3β inhibitor SB21 (5 μM) 1 hour prior to administration of elevated D-glucose significantly increased cell survival and decreased nuclear DNA fragmentation 48 hours following elevated glucose treatment (*p<0.01 vs. HG). Co-application of Wnt1 antibody (Wnt1Ab) did not alter the ability of SB21 to protect ECs during elevated glucose treatment (*P<0.01 vs. HG). (B and D) EPO (10 ng/ml) administered 1 hour prior to elevated D-glucose (25 mM) application significantly increased cell survival and decreased nuclear DNA fragmentation in ECs 48 hours following elevated glucose treatment (*p<0.01 vs. HG). Co-application of GSK-3β inhibitor SB21 with EPO significantly increased survival and decreased apoptotic nuclear DNA degradation during elevated glucose exposure, but lead to similar survival levels and DNA degradation during EPO administration alone with elevated glucose without a synergistic increase, suggesting that EPO requires the inhibition of GSK-3β activity for cytoprotection in ECs (*p<0.01 vs. HG alone). In all cases, each data point represents the mean and SEM and control = untreated EC cultures.

Article Snippet: For treatments applied 1 hour prior to elevated glucose concentrations, application of erythropoietin (EPO) (R&D Systems, Minneapolis, MN), human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN), a mouse monoclonal anti body against Wnt1 (R&D Systems, 0olis, MN), or the glycogen synthase kinase (GSK)-3β inhibitor SB216763 [3-(2,4-Dichlorophenyl)-4-(1-methyl- 1 H-indol-3-yl)- 1 H-pyrrole-2,5-dione] (SB21) (Tocris, Ellisville, MO) were continuous.

Techniques: Inhibition, Activity Assay, Control

Journal: Molecular Medicine Reports

Article Title: miR-144 promotes the proliferation and differentiation of bone mesenchymal stem cells by downregulating the expression of SFRP1

doi: 10.3892/mmr.2019.10252

Figure Lengend Snippet: Expression of miR-144, Sfrp1 and TNF-α in clinical samples. (A) Expression of miR-144 was detected by reverse transcription-quantitative polymerase chain reaction analysis. Serum levels of (B) Sfrp1 and (C) TNF-α were detected by ELISA. (D) Correlation analysis was performed to examine the relationship between miR-144 and Sfrp1. Data represent the mean ± SEM. **P<0.01 and ***P<0.001. OP, osteoporosis; miR, microRNA; Sfrp1, secreted frizzled-related protein 1.

Article Snippet: The testing kits for Sfrp1 (cat. no. CSB-E15074h) and TNF-α (cat. no. CSB-E04740h) were purchased from Cusabio Biotech Co., Ltd. (Wuhan, China).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Molecular Medicine Reports

Article Title: miR-144 promotes the proliferation and differentiation of bone mesenchymal stem cells by downregulating the expression of SFRP1

doi: 10.3892/mmr.2019.10252

Figure Lengend Snippet: Expression of Sfrp1 and Runx2 in BMSCs is regulated by miR-144. (A) Expression levels of miR-144, Sfrp1 and Runx2 were detected by reverse transcription-quantitative polymerase chain reaction analysis. (B) Protein expression of Sfrp1, Runx2, CDK4, Wnt1 and β-catenin was analyzed by western blot analysis. (C) Distributions of Sfrp1 and Runx2 in BMSCs were observed using an immunofluorescence assay (magnification, ×200). Data represent the mean ± SEM. **P<0.01 and ***P<0.001. miR, microRNA; BMSCs, bone marrow-derived mesenchymal stem cells; Sfrp1, secreted frizzled-related protein 1; Runx2, Runt-related transcription factor 2; CDK4, cyclin-dependent kinase 4.

Article Snippet: The testing kits for Sfrp1 (cat. no. CSB-E15074h) and TNF-α (cat. no. CSB-E04740h) were purchased from Cusabio Biotech Co., Ltd. (Wuhan, China).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Derivative Assay

Journal: Molecular Medicine Reports

Article Title: miR-144 promotes the proliferation and differentiation of bone mesenchymal stem cells by downregulating the expression of SFRP1

doi: 10.3892/mmr.2019.10252

Figure Lengend Snippet: miR-144 promotes the proliferation and differentiation of BMSCs by downregulating the expression of Sfrp1. (A) A luciferase reporter assay was used to examine the direct interaction between miR-144 and Sfrp1. The group of BMSCs were co-transfected with WT Sfrp1 plasmid and miR-144 mimic was compared with the other three groups. BMSCs were transfected with Sfrp1 siRNA, and the interference efficiency was analyzed by (B) reverse transcription-quantitative polymerase chain reaction and (C) western blot analysis, respectively. (D) A colony formation assay was used to determine how Sfrp1 affected the ability of miR-144 to promote BMSC proliferation, and (E) quantified. (F) Alizarin red staining was performed following 3 weeks of osteogenic differentiation to detect the calcium nodi in BMSCs (magnification, ×200), with (G) quantification of staining. (H) ALP activity assays were performed following 14 days of osteogenic differentiation. The group of BMSCs co-transfected with the miR-144 inhibitor and NC siRNA was compared with other two groups. (I) Protein expression levels of Sfrp1, Runx2, CDK4, Wnt1 and β-catenin were analyzed by western blot analysis. GAPDH was used as an internal control. Data represent the mean ± SEM. **P<0.01 and ***P<0.001. miR, microRNA; BMSCs, bone marrow-derived mesenchymal stem cells; WT, wild-type; MUT, mutant; siRNA, small interfering RNA; NC, negative control; ALP, alkaline phosphatase; Sfrp1, secreted frizzled-related protein 1; Runx2, Runt-related transcription factor 2; CDK4, cyclin-dependent kinase 4.

Article Snippet: The testing kits for Sfrp1 (cat. no. CSB-E15074h) and TNF-α (cat. no. CSB-E04740h) were purchased from Cusabio Biotech Co., Ltd. (Wuhan, China).

Techniques: Expressing, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Colony Assay, Staining, Activity Assay, Control, Derivative Assay, Mutagenesis, Small Interfering RNA, Negative Control

Journal: Journal of Biological Chemistry

Article Title: A WNT/β-Catenin Signaling Activator, R-spondin, Plays Positive Regulatory Roles during Skeletal Myogenesis

doi: 10.1074/jbc.m110.169391

Figure Lengend Snippet: FIGURE 5. Activation of WNT/-catenin signaling and WNT/-catenin-dependent MYF5 induction by RSPO2. A, dose-dependent activation of a WNT/- catenin signaling reporter, sTopFlash, by the RSPO2 protein in undifferentiated C2C12 cells. sTopFlash reporter DNA construct was transiently transfected into C2C12 cells, and the cells were stimulated with various concentrations of the RSPO2 protein as well as WNT3A-conditioned medium (CM) for 24 h. Reporter luciferase activity was measured and normalized by the activity of cotransfected control Renilla luciferase construct. B, stabilization of the cytoplasmic -catenin protein by RSPO2 in differentiating C2C12 cells. C2C12 cells were differentiated for up to 4 days (D1–D4) in the absence (BSA; 200 ng/ml) or presence of the Rspo2 protein (200 ng/ml). The RSPO2 protein or BSA was added daily. Cytoplasmic fractions were prepared and analyzed for -catenin and -tubulin protein expression by Western blot. C, C2C12 cells were transiently transfected with sTopFlash reporter and stimulated with the RSPO2 protein (200 ng/ml) for 24 h. WNT signaling antagonists, the DKK1 and sFRP1 proteins, were simultaneously added at the indicated concentrations (ng/ml). sTopFlash activities were normalized by Renilla control luciferase activities. D, MYF5 protein expression was analyzed in C2C12 cells costimulated with the RSPO2 (200 ng/ml) and DKK1 or sFRP1 proteins by Western blot. E, RSPO2-induced sTopFlash reporter activities were measured in C2C12 cells transiently transfected with a dominant- negative human TCF4 construct (NTCF4-Myc). F, MYF5 expression in C2C12 cells stably expressing NTCF4. Expression of NTCF4 was determined using anti-MYC antibody by Western blot. Error bars are presented as S.E.

Article Snippet: Recombinant RSPO2, DKK1, and sFRP1 proteins were purchased from R&D Systems (Minneapolis, MN) and used at the indicated concentrations.

Techniques: Activation Assay, Construct, Transfection, Luciferase, Activity Assay, Control, Expressing, Western Blot, Dominant Negative Mutation, Stable Transfection