Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-503-3p affects osteogenic differentiation of human adipose-derived stem cells by regulation of Wnt2 and Wnt7b under cyclic strain

doi: 10.1186/s13287-020-01842-0

Figure Lengend Snippet: The sequence of primer used for RT-PCR in this study

Article Snippet: The primary antibodies used in western blot were listed as follows: RUNX2 (Abcam, USA), ALP (Abcam, USA), SPARC (Abcam, USA), Wnt7b (R&D Systems, USA), Wnt2 (R&D Systems, USA), GAPDH (Cell Signaling Technology, USA), and β-catenin (Abcam, USA) and followed by washing of membranes thrice in TBS-0.05% Tween 20, followed by incubation at room temperature with the corresponding secondary antibodies for 1 h. Blots were then incubated in the dark with ECL (Thermo Fisher Scientific, Germany) and visualized by exposing to enhanced chemiluminescence reagents (GE Healthcare, USA).

Techniques: Sequencing

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-503-3p affects osteogenic differentiation of human adipose-derived stem cells by regulation of Wnt2 and Wnt7b under cyclic strain

doi: 10.1186/s13287-020-01842-0

Figure Lengend Snippet: Wnt2 and Wnt7b were targets of miR-503-3p. (* p < 0.05, there were significant differences between these two groups) a The sequences of miR-503-3p are highly conservative across species. b miR-503-3p might bind to Wnt2 3′-UTR. c miR-503-3p might bind to Wnt7b 3′-UTR. d The luciferase activity of hASCs co-transfected with miR-503-3p mimic and wt-Wnt2 significantly reduced, compared to the miR-Ctrl mimic and wt-Wnt2 group. The luciferase activity of hASCs co-transfected with miR-503-3p mimic and mut-Wnt2 had no significance, compared to miR-Ctrl mimic and mut-Wnt2 group. e The luciferase activity of hASCs co-transfected with miR-503-3p mimic and wt-Wnt7b significantly reduced, compared to the miR-Ctrl mimic and wt-Wnt7b group. The luciferase activity of hASCs co-transfected with miR-503-3p mimic and mut-Wnt7b had no significance, compared to miR-Ctrl mimic and mut-Wnt7b group. f Compared to the miR-Ctrl mimic group, Wnt2 mRNA levels decreased in hASCs transfected with miR-503-3p mimic as shown by real-time PCR. g Compared to the miR-Ctrl mimic group, Wnt7b mRNA levels decreased in hASCs transfected with miR-503-3p mimic as shown by real-time PCR. e Protein blots were listed in the upper column. Compared to the miR-Ctrl mimic group, Wnt2 protein levels decreased in hASCs transfected miR-503-3p mimic as shown by Western blot. f Protein blots were listed in the upper column. Compared to the miR-Ctrl mimic group, Wnt7b protein levels decreased in hASCs transfected miR-503-3p mimic as shown by western blot

Article Snippet: The primary antibodies used in western blot were listed as follows: RUNX2 (Abcam, USA), ALP (Abcam, USA), SPARC (Abcam, USA), Wnt7b (R&D Systems, USA), Wnt2 (R&D Systems, USA), GAPDH (Cell Signaling Technology, USA), and β-catenin (Abcam, USA) and followed by washing of membranes thrice in TBS-0.05% Tween 20, followed by incubation at room temperature with the corresponding secondary antibodies for 1 h. Blots were then incubated in the dark with ECL (Thermo Fisher Scientific, Germany) and visualized by exposing to enhanced chemiluminescence reagents (GE Healthcare, USA).

Techniques: Luciferase, Activity Assay, Transfection, Real-time Polymerase Chain Reaction, Western Blot

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-503-3p affects osteogenic differentiation of human adipose-derived stem cells by regulation of Wnt2 and Wnt7b under cyclic strain

doi: 10.1186/s13287-020-01842-0

Figure Lengend Snippet: Effect of Wnt2 on osteogenic differentiation of hASCs under cyclic strain. (* p < 0.05, there were significant differences between these two groups) a The results of real-time PCR showed that the Wnt2b expression in EX-Wnt2b group increased compared to the EX-Ctrl group; Wnt2 expression in the siWnt2 group significantly decreased, compared to the siR-Ctrl group. b Protein blots were listed in the left column. The results of western blot showed that Wnt2 expression in the EX-Wnt2 group increased, compared to the EX-Ctrl group; Wnt2 expression in the siWnt2 group significantly decreased, compared to the siR-Ctrl group. c After EX-Wnt2 transfection, the results of real-time PCR showed that RUNX2, ALP, SPARC, Wnt7b, and β-catenin significantly enhanced, compared to the EX-Ctrl group. d After siWnt2 transfection, the results of real-time PCR showed that RUNX2, ALP, SPARC, Wnt7b, and β-catenin significantly decreased, compared to the siR-Ctrl group. e Comparing to the EX-Ctrl group, both the ALP activity and the content of Ca 2+ were significantly increased when EX-Wnt2 transfected. f Comparing to the siR-Ctrl group, both the ALP activity and the content of Ca 2+ were significantly decreased when siWnt2 transfected

Article Snippet: The primary antibodies used in western blot were listed as follows: RUNX2 (Abcam, USA), ALP (Abcam, USA), SPARC (Abcam, USA), Wnt7b (R&D Systems, USA), Wnt2 (R&D Systems, USA), GAPDH (Cell Signaling Technology, USA), and β-catenin (Abcam, USA) and followed by washing of membranes thrice in TBS-0.05% Tween 20, followed by incubation at room temperature with the corresponding secondary antibodies for 1 h. Blots were then incubated in the dark with ECL (Thermo Fisher Scientific, Germany) and visualized by exposing to enhanced chemiluminescence reagents (GE Healthcare, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Activity Assay

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-503-3p affects osteogenic differentiation of human adipose-derived stem cells by regulation of Wnt2 and Wnt7b under cyclic strain

doi: 10.1186/s13287-020-01842-0

Figure Lengend Snippet: Effect of Wnt7b on osteogenic differentiation of hASCs under cyclic strain. (* p < 0.05, there were significant differences between these two groups) a The results of real-time PCR showed that the Wnt7b expression in EX-Wnt7b group increased, compared to the EX-Ctrl group; Wnt7b expression in the siWnt7b group significantly decreased, compared to the siR-Ctrl group. b Protein blots were listed in the left column. The results of western blot showed that Wnt7b expression in the EX-Wnt7b group increased, compared to the EX-Ctrl group; Wnt7b expression in the siWnt7b group significantly decreased, compared to the siR-Ctrl group. c After EX-Wnt7b transfection, the results of real-time PCR showed that RUNX2, ALP, SPARC, Wnt7b, and β-catenin significantly enhanced, compared to the EX-Ctrl group. d After siWnt7b transfection, the results of real-time PCR showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, and β-catenin significantly decreased, compared to the siR-Ctrl group. e Comparing to the EX-Ctrl group, ALP activity and the content of Ca 2+ significantly increased when EX-Wnt7b transfected. Comparing to the siR-Ctrl group, ALP activity and the content of Ca 2+ significantly decreased when siWnt7b transfected

Article Snippet: The primary antibodies used in western blot were listed as follows: RUNX2 (Abcam, USA), ALP (Abcam, USA), SPARC (Abcam, USA), Wnt7b (R&D Systems, USA), Wnt2 (R&D Systems, USA), GAPDH (Cell Signaling Technology, USA), and β-catenin (Abcam, USA) and followed by washing of membranes thrice in TBS-0.05% Tween 20, followed by incubation at room temperature with the corresponding secondary antibodies for 1 h. Blots were then incubated in the dark with ECL (Thermo Fisher Scientific, Germany) and visualized by exposing to enhanced chemiluminescence reagents (GE Healthcare, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Activity Assay

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-503-3p affects osteogenic differentiation of human adipose-derived stem cells by regulation of Wnt2 and Wnt7b under cyclic strain

doi: 10.1186/s13287-020-01842-0

Figure Lengend Snippet: Effect of β-catenin on osteogenic differentiation of hASCs under cyclic strain. (* p < 0.05, there were significant differences between these two groups) a The results of real-time PCR showed that the β-catenin expression in EX-β-catenin group increased, compared to the EX-Ctrl group; β-catenin expression in the siβ-catenin group significantly decreased, compared to the siR-Ctrl group. b Protein blots were listed in the left column. The results of western blot showed that cytoplasmic β-catenin and nuclear β-catenin expression in the EX-β-catenin group increased, compared to the EX-Ctrl group; cytoplasmic β-catenin and nuclear β-catenin expression in the siβ-catenin group significantly decreased, compared to the siR-Ctrl group. c After EX-β-catenin transfection, the results of real-time PCR showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, and β-catenin significantly enhanced, compared to the EX-Ctrl group. d After siβ-catenin transfection, the results of real-time PCR showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, and β-catenin significantly decreased, compared to the siR-Ctrl group. e Comparing to the EX-Ctrl group, ALP activity and the content of Ca 2+ significantly increased when EX-β-catenin transfected. Comparing to the siR-Ctrl group, ALP activity and the content of Ca 2+ significantly decreased when siβ-catenin transfected

Article Snippet: The primary antibodies used in western blot were listed as follows: RUNX2 (Abcam, USA), ALP (Abcam, USA), SPARC (Abcam, USA), Wnt7b (R&D Systems, USA), Wnt2 (R&D Systems, USA), GAPDH (Cell Signaling Technology, USA), and β-catenin (Abcam, USA) and followed by washing of membranes thrice in TBS-0.05% Tween 20, followed by incubation at room temperature with the corresponding secondary antibodies for 1 h. Blots were then incubated in the dark with ECL (Thermo Fisher Scientific, Germany) and visualized by exposing to enhanced chemiluminescence reagents (GE Healthcare, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Activity Assay

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-503-3p affects osteogenic differentiation of human adipose-derived stem cells by regulation of Wnt2 and Wnt7b under cyclic strain

doi: 10.1186/s13287-020-01842-0

Figure Lengend Snippet: Effects of miR-503-3p overexpression and inhibition on osteogenic differentiation of hASCs under cyclic strain. (* p < 0.05, there were significant differences between these two groups) a The expression of miR-503-3p in hASCs transfected with miR-503-3p mimic markedly increased, compared to the miR-Ctrl mimic group. b The expression of miR-503-3p in hASCs transfected with miR-503-3p inhibitor decreased, compared to the miR-Ctrl inhibitor group. c After miR-503-3p mimic transfection, the results of real-time PCR showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, and β-catenin decreased, compared to the miR-Ctrl mimic group. d Protein blots were listed in the left column. After miR-503-3p mimic transfection, the results of western blot showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, cytoplasmic β-catenin, and nuclear β-catenin decreased. e Both ALP activity and the content of Ca 2+ significantly decreased when miR-503-3p mimic transfected. f After miR-503-3p mimic transfection, the immunofluorescence intensity of β-catenin in the hASC nucleus was decreased. g After miR-503-3p inhibitor transfection, the results of real-time PCR showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, and β-catenin increased, compared to the miR-Ctrl inhibitor group. h Protein blots were listed in the left column. After miR-503-3p inhibitor transfection, the results of western blot showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, cytoplasmic β-catenin, and nuclear β-catenin decreased, compared to the miR-Ctrl inhibitor group. i Both ALP activity and the content of Ca 2+ significantly increased when miR-503-3p inhibitor transfected. j After miR-503-3p inhibitor transfection, the immunofluorescence intensity of β-catenin in the hASC nucleus was increased

Article Snippet: The primary antibodies used in western blot were listed as follows: RUNX2 (Abcam, USA), ALP (Abcam, USA), SPARC (Abcam, USA), Wnt7b (R&D Systems, USA), Wnt2 (R&D Systems, USA), GAPDH (Cell Signaling Technology, USA), and β-catenin (Abcam, USA) and followed by washing of membranes thrice in TBS-0.05% Tween 20, followed by incubation at room temperature with the corresponding secondary antibodies for 1 h. Blots were then incubated in the dark with ECL (Thermo Fisher Scientific, Germany) and visualized by exposing to enhanced chemiluminescence reagents (GE Healthcare, USA).

Techniques: Over Expression, Inhibition, Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Immunofluorescence

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-503-3p affects osteogenic differentiation of human adipose-derived stem cells by regulation of Wnt2 and Wnt7b under cyclic strain

doi: 10.1186/s13287-020-01842-0

Figure Lengend Snippet: Effect of Wnt2 and Wnt7b regulated by miR-503-3p on osteogenic differentiation of hASCs under cyclic strain (* p < 0.05, there were significant differences between these two groups) a After co-transfecting miR-503-3p mimic with EX-Wnt2 and EX-Wnt7b, real-time PCR showed RUNX2, ALP, SPARC, Wnt2, Wnt7b, and β-catenin were increased, than the miR-503-3p mimic and EX-Ctrl group. b Protein blots were listed in the left column. After co-transfecting miR-503-3p mimic with EX-Wnt2 and EX-Wnt7b, western blot showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, cytoplasmic, and nuclear β-catenin increased, than the miR-503-3p mimic and EX-Ctrl group. c Both ALP activity and the content of Ca 2+ significantly increased when co-transfecting miR-503-3p mimic with EX-Wnt2 and EX-Wnt7b. d After co-transfecting miR-503-3p mimic with EX-Wnt2 and EX-Wnt7b, the immunofluorescence intensity of nucleus β-catenin was enhanced. e After co-transfecting miR-503-3p inhibitor with siWnt2 and siWnt7b, real-time PCR showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, and β-catenin decreased, than the miR-503-3p inhibitor and siR-Ctrl group. f Protein blots were listed in the left column. After co-transfecting miR-503-3p inhibitor with siWnt2 and siWnt7b, western blot showed RUNX2, ALP, SPARC, Wnt2, Wnt7b, cytoplasmic, and nuclear β-catenin decreased, than the miR-503-3p inhibitor and siR-Ctrl group. g Both ALP activity and the content of Ca 2+ significantly decreased when co-transfecting miR-503-3p inhibitor with siWnt2 and siWnt7b. h After co-transfecting miR-503-3p inhibitor with siWnt2 and siWnt7b, the immunofluorescence intensity of nucleus β-catenin was decreased. i After co-transfecting miR-503-3p inhibitor with siβ-catenin, real-time PCR showed that RUNX2, ALP, and SPARC decreased; Wnt2, Wnt7b and β-catenin decreased, than the miR-503-3p inhibitor and siR-Ctrl group. j Protein blots were listed in the left column. After co-transfecting miR-503-3p inhibitor with siβ-catenin, western blot showed that RUNX2, ALP, and SPARC decreased; Wnt2, Wnt7b, cytoplasmic and nuclear β-catenin decreased, than the miR-503-3p inhibitor and siR-Ctrl group. k Both ALP activity and the content of Ca 2+ significantly decreased when co-transfecting miR-503-3p inhibitor with siβ-catenin. l After co-transfecting miR-503-3p inhibitor with siβ-catenin into hASCs, the immunofluorescence intensity of nucleus β-catenin was decreased. m After co-transfecting miR-503-3p mimic with EX-β-catenin, real-time PCR showed that RUNX2, ALP, and SPARC increased; Wnt2, Wnt7b, and β-catenin increased, than the miR-503-3p mimic and EX-Ctrl group. n Protein blots were listed in the left column. After co-transfecting miR-503-3p mimic with EX-β-catenin, western blot showed that RUNX2, ALP, and SPARC increased; Wnt2, Wnt7b, cytoplasmic, and nuclear β-catenin increased, than the miR-503-3p mimic and EX-Ctrl group. o Both ALP activity and the content of Ca 2+ significantly increased when co-transfecting miR-503-3p mimic with EX-β-catenin. p After co-transfecting miR-503-3p mimic with EX-β-catenin, the immunofluorescence intensity of nucleus β-catenin was increased

Article Snippet: The primary antibodies used in western blot were listed as follows: RUNX2 (Abcam, USA), ALP (Abcam, USA), SPARC (Abcam, USA), Wnt7b (R&D Systems, USA), Wnt2 (R&D Systems, USA), GAPDH (Cell Signaling Technology, USA), and β-catenin (Abcam, USA) and followed by washing of membranes thrice in TBS-0.05% Tween 20, followed by incubation at room temperature with the corresponding secondary antibodies for 1 h. Blots were then incubated in the dark with ECL (Thermo Fisher Scientific, Germany) and visualized by exposing to enhanced chemiluminescence reagents (GE Healthcare, USA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Immunofluorescence

Journal: Cancer Cell International

Article Title: Identification of the prognostic value of a 2-gene signature of the WNT gene family in UCEC using bioinformatics and real-world data

doi: 10.1186/s12935-021-02215-0

Figure Lengend Snippet: Expression of WNT2 and WNT10A in UCEC. A – B Representativeness of weak WNT2 staining in UCEC (200× and 400×); C – D Representativeness of moderate WNT2 staining in UCEC (200× and 400×); E – F Representativeness of strong WNT2 staining in UCEC (200× and 400×); G – H Representativeness of weak WNT10A staining in UCEC (200× and 400×); I – J Representativeness of moderate WNT10A staining in UCEC (200× and 400×); K – L Representativeness of strong WNT10A staining in UCEC (200× and 400×)

Article Snippet: Rabbit anti-human WNT2 polyclonal antibody was purchased from Solarbio (Beijing, China).

Techniques: Expressing, Staining

Journal: World Journal of Gastroenterology

Article Title: Down-regulation of miR-30a-3p/5p promotes esophageal squamous cell carcinoma cell proliferation by activating the Wnt signaling pathway

doi: 10.3748/wjg.v23.i45.7965

Figure Lengend Snippet: miR-30a-3p/5p directly target the 3'-UTRs of Wnt2 and Fzd2, respectively. A: Target gene screening of miR-30a-3p (left) and miR-30a-5p (right) by qPCR suggested that miR-30a-3p targeted Wnt2 and miR-30a-5p targeted Fzd2 in the Wnt signaling pathway; B: Predicted target sequences of miR-30a-3p (top) and miR-30a-5p (bottom) in the 3’-UTRs of WNT2 and FZD2, respectively; C: qPCR (top) and Western blot (bottom), respectively, confirmed the inhibitory effects of miR-30a-3p and miR-30a-5p on Wnt2 and Fzd2 at both mRNA and protein levels. GAPDH and α-tubulin were, respectively, used as loading controls for qPCR and Western blot; D: Luciferase reporter assay indicated that miR-30-3p and miR-30a-5p directly targeted the wild-type 3’-UTRs of Wnt2 and Fzd2, respectively, but had no effect on the mutant ones. Two concentrations of miR-30a-3p- and miR-30a-5p-mimics (10 nmol/L and 20 nmol/L) plus wild-type or mutant 3’-UTRs of target genes were applied. Error bars represent the mean ± SD of three independent experiments; a P < 0.05.

Article Snippet: Proteins were isolated, subjected to SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes, and incubated with anti-Cyclin D1 (Abcam, Cambridge, MA, United States), anti-p27 (Abcam, Cambridge, MA, United States), anti-p21 (Abcam, Cambridge, MA, United States), anti-WNT2 (Bioworld Technology Inc. St. Louis Park, MN, United States), and anti-FZD2 (Bioworld Technology Inc. St. Louis Park, MN, United States) antibodies. α-tubulin (Sigma-Aldrich; Merck Millipore) was used as a loading control.

Techniques: Western Blot, Luciferase, Reporter Assay, Mutagenesis