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: Journal of Molecular Endocrinology

Article Title: Glucocorticoid inhibits cell proliferation in differentiating osteoblasts by microRNA-199a targeting of WNT signaling

doi: 10.1530/jme-14-0314

Figure Lengend Snippet: Figure 8. Repression of WNT signaling by miR-199a-5p transfection. A: Western blot analysis of FZD4 and WNT2 expression in primary osteoblasts treated with 10-7 M Dex for 5 days. n = 3, **P < 0.01. B: Western

Article Snippet: After blocking with 0.1% Tween 20 and 5% nonfat dry milk in 11 Tris-buffered saline at room temperature for 1 h, the membrane was incubated overnight at 4°C in 12 one of the following primary antibodies: FZD4 (Peprotech, Rocky Hill, NJ) (1:400) , WNT2 (Santa 13 Cruz, CA, USA) (1:400), B-catenin (Santa Cruz, CA, USA) (1:200), Runx2 (Santa Cruz, CA, USA) 14 (1:200), Osterix (Santa Cruz, CA, USA) (1:400) and β-actin (Santa Cruz, CA, USA) (1:1000) as an 15 internal control.

Techniques: Transfection, Western Blot, Expressing

Journal: bioRxiv

Article Title: Fgf9-Nolz-1-Wnt2 Signaling Axis Regulates Morphogenesis of the Lung

doi: 10.1101/2022.08.10.503529

Figure Lengend Snippet: (A) Morphogenic molecules are screened by qRT-PCR in E12.5 Nolz-1 mutant lungs. Wnt2 and Lef1 mRNA are decreased in Nolz-1 mutant lungs. Fgf10 is decreased and Pdgfrα is increased in the Nolz-1 mutant lungs. Gli3 and Bmp4 are decreased and Tgf-β2 is increased in Nolz-1 mutant lungs. Student’s t- test, * P < 0.05, ** P < 0.01, n = 3. (B) In situ hybridization shows that Wnt2 mRNA is mainly expressed in the distal parts of the mesenchyme of E11.5 and E13.5 wild type lungs. Wnt2 mRNA is markedly decreased in mutant mesenchyme. The insets show high magnification of the regions indicated by asterisks. Scale bars, 50 μm. (C) The qRT-PCR assay shows that over-expression of Nolz-1 by electroporation of pcBIG-myc-Nolz-1-ires-EGFP plasmid up-regulates Wnt2 , cyclinD1 and c-myc mRNAs in primary mesenchymal cell culture derived from E14.5 wild type mouse lungs compared to mock transfection of pcBIG-ires-EGFP control plasmids. Student’s t- test, * P < 0.05, *** P < 0.001, n = 4. (D) The schematic drawing illustrates the locations of the putative Nolz-1 binding sites of “AGGAT” at -788 (N1 motif) and -2194 (N2 motif) in 5 ‘flânking regions of mouse Wnt2 gene (+1: ATG translation start site). (E) The chromatin immunoprecipitation (ChIP) assay shows that a 187 bp PCR band is amplified from the N2 locus with the immunoprecipitated products using the anti-myc antibody, but not the control rabbit IgG, in E14.5 lung mesenchymal cell culture electroporated with pcBIG-myc-Nolz-1-ires-EGFP plasmids. No specific PCR band is detected from the N1 locus. Student’s t- test, * P < 0.05, n.s. not significant, n = 3. (F) The reporter gene assay showed that the luciferase activity is increased in the pGL3-N2-Luc group compared to the pGL3-Luc control group in E14.5 lung mesenchymal cells transfected with pcBIG-myc-Nolz-1-ires-EGFP plasmids. Student’s t- test, ** P < 0.01, n = 6.

Article Snippet: The lung explants were cultured with recombinant human Wnt2 protein (150 ng/ml, Novus Biologicals).

Techniques: Quantitative RT-PCR, Mutagenesis, In Situ Hybridization, Over Expression, Electroporation, Plasmid Preparation, Cell Culture, Derivative Assay, Transfection, Control, Binding Assay, Chromatin Immunoprecipitation, Amplification, Immunoprecipitation, Reporter Gene Assay, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Fgf9-Nolz-1-Wnt2 Signaling Axis Regulates Morphogenesis of the Lung

doi: 10.1101/2022.08.10.503529

Figure Lengend Snippet: (A) Treatment with rFgf10 (200 n /ml) of wild type explant lung culture for 48 hr. qRT-PCR shows that Nolz-1 and Wnt2 are not changed in the rFgf10 treated group compared to the vehicle control. Student’s t -test, P > 0.05, n = 4. Scale bar, 500 μm. (B) Treatment with rFgf9 (200 ng/ml) results in enlarged epithelia in wild-type explant lungs cultured for 48 hr. The qRT-PCR shows that Nolz-1 , Wnt2 and Lef1 mRNAs are increased in rFgf9 treated group in wild type lungs. Student’s t -test, * P < 0.05, ** P < 0.01, n = 4. Western blotting showed that rFgf9 treatment increases Nolz-1 protein by 99% in wild type lung culture compared to the vehicle-treated group. Student’s t -test, * P < 0.05, n = 3. (C) Working hypothesis. Nolz-1 controls the proliferation of mesenchymal cells and the growth of epithelial branches through the regulation of Wnt2 signaling in the early stages of the development of the lungs. In the late stages of development, Nolz-1 acts non-cell autonomously to regulate the development of epithelial cells through Wnt2 signaling. Fgf9 acts upstream to regulate Nolz-1 expression in developing lungs.

Article Snippet: The lung explants were cultured with recombinant human Wnt2 protein (150 ng/ml, Novus Biologicals).

Techniques: Quantitative RT-PCR, Control, Cell Culture, Western Blot, Expressing

Journal: Scientific reports

Article Title: Gene Expression Signatures Point to a Male Sex-Specific Lung Mesenchymal Cell PDGF Receptor Signaling Defect in Infants Developing Bronchopulmonary Dysplasia.

doi: 10.1038/s41598-018-35256-z

Figure Lengend Snippet: Figure 2. Validation of the differential gene expression between MSCs from male infants developing BPD and combined controls. mRNA expression was assessed by quantitative PCR and protein expression was assessed by immunoblotting and ELISA. (A) Compared to the control group MSCs from male infants developing BPD showed significantly lower expression of PDGFRA, FGF7, WNT2 and MMP3 and a trend for lower SPRY1 and FOXF2 mRNA expression. (B) Representative immunoblot confirms decreased protein expression of PDGFRα. MSCs from 5 male infants developing BPD and 4 control infants (one male and one female infants who were not developing BPD and two female infants developing BPD) are shown. Full-length blots are available in Supplemental Fig. S1. (C) PDGFRα densitometry analysis group mean data for 9 male infants developing BPD and 18 controls. (D) MSCs from male infants developing BPD secrete significantly lower concentrations of FGF-7, MMP3 and Wnt2. Data are means ± SE. Statistical significance was determined by unpaired t-test.

Article Snippet: Protein levels of FGF7, Wnt2 and MMP3 in MSC supernatants were measured by enzyme-linked immunosorbent assay (FGF7 and MMP3 from R&D Systems, Minneapolis, MN and Wnt2 from CUSABIO Biotech, China).

Techniques: Biomarker Discovery, Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Scientific reports

Article Title: Gene Expression Signatures Point to a Male Sex-Specific Lung Mesenchymal Cell PDGF Receptor Signaling Defect in Infants Developing Bronchopulmonary Dysplasia.

doi: 10.1038/s41598-018-35256-z

Figure Lengend Snippet: Figure 3. Correlation analysis of lung development gene expression levels. The relationship between the mRNA expression levels of PDGFRA, FGF7, WNT2, SPRY1 and FOXF2 was analyzed by Pearson correlation analysis.

Article Snippet: Protein levels of FGF7, Wnt2 and MMP3 in MSC supernatants were measured by enzyme-linked immunosorbent assay (FGF7 and MMP3 from R&D Systems, Minneapolis, MN and Wnt2 from CUSABIO Biotech, China).

Techniques: Gene Expression, Expressing

Journal: Science advances

Article Title: Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

doi: 10.1126/sciadv.ade3816

Figure Lengend Snippet: Fig. 3. RNA sequencing reveals WNT2 up-regulation upon sotorasib treatment. Volcano plot representing gene expression changes between (A) H23 parental cells, sotorasib treated versus untreated, and (B) isogenic resistant H23 cells compared to parental cells. Statistical significance was calculated for control verse treatment. n = 3 per group. (C) Number of overlapping and unique genes that were up-regulated or down-regulated with respect to treatment represented as a Venn diagram. (D) Heatmap representing top 10 overlapping up-regulated and down-regulated genes based on average fold change. (E and F) Effect of 3.2 μM sotorasib on H23 parental or isogenic resistant (Iso R) cells having knockdown of ITGB4 or CTNNB1 or both represented as percent change in growth at 96 hours (bar graph), respectively. Two-way ANOVA was used to calculate the statistical significance for each time point and for each condition (si Control, si ITGB4, si CTNNB1, si CTNNB1 + si ITGB4; n = 3 per group; ****P < 0.0001. (G) Immunoblot confirmed knockdown of ITGB4 and β-catenin in H23 parental cells and H23 sotorasib (20 μM) resistant cells. These cells were also treated with 3.2 μM sotorasib for 72 hours to identify changes in protein expression and signaling. (H) Representing the effect of ITGB4 and CTNNB1 single knockdown or double knockdown together with 10 μM sotorasib as a percent change in growth. Two-way ANOVA was used to calculate the statistical significance for each time point and each condition (si Control, si PXN, si ITGB4, and si PXN + si ITGB4; n = 3 per group; ****P < 0.0001. (I) Immunoblot showing knockdown of ITGB4 and β-catenin and changes in expression of active β-catenin, γH2AX, and p27 in SW1573 cells. (J) Immunoblot showing the reduction in the expression of WNT2, ITGB4, phospho, and total β-catenin in the SW1573 treated with sotorasib and CFZ drug combination.

Article Snippet: We first generated a WNT2 knockout (KO) H23 cell line using the combination of WNT2 CRISPR-Cas9 KO and WNT2 homology directed repair (HDR) plasmids from Santa Cruz Biotechnologies.

Techniques: RNA Sequencing, Gene Expression, Control, Knockdown, Western Blot, Expressing

Journal: Science advances

Article Title: Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

doi: 10.1126/sciadv.ade3816

Figure Lengend Snippet: Fig. 5. KRAS G12C inhibitors have a differential effect on cell growth and progression. (A) Effect of increasing concentrations (1 to 4 μM) of sotorasib and adagrasib exhibited different effects on cell proliferation over 24 hours in H23 cells. Two-way ANOVA was used for calculating statistical significance across various time points and for each drug concentration. n = 3. (B) Effect of KRAS inhibitors sotorasib or adagrasib increasing concentrations (1 to 8 μM) on SW1573 cells within 24 hours of drug treatment. Two-way ANOVA test was used to calculate the statistical significance. n = 3 sample per group. (C) Immunofluorescence image of SW1573 to support the dose dependent inhibitory effect of adagrasib on cell growth and expression of WNT2 (green) and phospsho-S675–β-catenin (magenta). The region of interest was zoomed 50% digitally to show membrane blebbing induced by adagrasib (white arrows). (D and E) Cell cycle dynamics were determined using IncuCyte Cell Cycle Lentivirus Reagent with fluorescence indicating cell cycle phase (brightfield image and schematic). Effect of increasing concentrations of sotorasib (1.25 to 20 μM) and adagrasib (0.6 to 1.25 μM) on cell cycle in SW1573 cells represented as pseudo color plots. The y axis of the plot represents events positive for GFP, and x axis represents the events positive for mKate2. mKate positive represents G1; GFP positive represents S, G2, and M; and double positive represents G1-S–transitioning cells.

Article Snippet: We first generated a WNT2 knockout (KO) H23 cell line using the combination of WNT2 CRISPR-Cas9 KO and WNT2 homology directed repair (HDR) plasmids from Santa Cruz Biotechnologies.

Techniques: Concentration Assay, Immunofluorescence, Expressing, Membrane, Fluorescence

Journal: Science advances

Article Title: Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

doi: 10.1126/sciadv.ade3816

Figure Lengend Snippet: Fig. 3. RNA sequencing reveals WNT2 up-regulation upon sotorasib treatment. Volcano plot representing gene expression changes between (A) H23 parental cells, sotorasib treated versus untreated, and (B) isogenic resistant H23 cells compared to parental cells. Statistical significance was calculated for control verse treatment. n = 3 per group. (C) Number of overlapping and unique genes that were up-regulated or down-regulated with respect to treatment represented as a Venn diagram. (D) Heatmap representing top 10 overlapping up-regulated and down-regulated genes based on average fold change. (E and F) Effect of 3.2 μM sotorasib on H23 parental or isogenic resistant (Iso R) cells having knockdown of ITGB4 or CTNNB1 or both represented as percent change in growth at 96 hours (bar graph), respectively. Two-way ANOVA was used to calculate the statistical significance for each time point and for each condition (si Control, si ITGB4, si CTNNB1, si CTNNB1 + si ITGB4; n = 3 per group; ****P < 0.0001. (G) Immunoblot confirmed knockdown of ITGB4 and β-catenin in H23 parental cells and H23 sotorasib (20 μM) resistant cells. These cells were also treated with 3.2 μM sotorasib for 72 hours to identify changes in protein expression and signaling. (H) Representing the effect of ITGB4 and CTNNB1 single knockdown or double knockdown together with 10 μM sotorasib as a percent change in growth. Two-way ANOVA was used to calculate the statistical significance for each time point and each condition (si Control, si PXN, si ITGB4, and si PXN + si ITGB4; n = 3 per group; ****P < 0.0001. (I) Immunoblot showing knockdown of ITGB4 and β-catenin and changes in expression of active β-catenin, γH2AX, and p27 in SW1573 cells. (J) Immunoblot showing the reduction in the expression of WNT2, ITGB4, phospho, and total β-catenin in the SW1573 treated with sotorasib and CFZ drug combination.

Article Snippet: We first generated a WNT2 knockout (KO) H23 cell line using the combination of WNT2 CRISPR-Cas9 KO and WNT2 homology directed repair (HDR) plasmids from Santa Cruz Biotechnologies.

Techniques: RNA Sequencing, Gene Expression, Control, Knockdown, Western Blot, Expressing

Journal: Science advances

Article Title: Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

doi: 10.1126/sciadv.ade3816

Figure Lengend Snippet: Fig. 5. KRAS G12C inhibitors have a differential effect on cell growth and progression. (A) Effect of increasing concentrations (1 to 4 μM) of sotorasib and adagrasib exhibited different effects on cell proliferation over 24 hours in H23 cells. Two-way ANOVA was used for calculating statistical significance across various time points and for each drug concentration. n = 3. (B) Effect of KRAS inhibitors sotorasib or adagrasib increasing concentrations (1 to 8 μM) on SW1573 cells within 24 hours of drug treatment. Two-way ANOVA test was used to calculate the statistical significance. n = 3 sample per group. (C) Immunofluorescence image of SW1573 to support the dose dependent inhibitory effect of adagrasib on cell growth and expression of WNT2 (green) and phospsho-S675–β-catenin (magenta). The region of interest was zoomed 50% digitally to show membrane blebbing induced by adagrasib (white arrows). (D and E) Cell cycle dynamics were determined using IncuCyte Cell Cycle Lentivirus Reagent with fluorescence indicating cell cycle phase (brightfield image and schematic). Effect of increasing concentrations of sotorasib (1.25 to 20 μM) and adagrasib (0.6 to 1.25 μM) on cell cycle in SW1573 cells represented as pseudo color plots. The y axis of the plot represents events positive for GFP, and x axis represents the events positive for mKate2. mKate positive represents G1; GFP positive represents S, G2, and M; and double positive represents G1-S–transitioning cells.

Article Snippet: We first generated a WNT2 knockout (KO) H23 cell line using the combination of WNT2 CRISPR-Cas9 KO and WNT2 homology directed repair (HDR) plasmids from Santa Cruz Biotechnologies.

Techniques: Concentration Assay, Immunofluorescence, Expressing, Membrane, Fluorescence