Journal: Frontiers in Cell and Developmental Biology

Article Title: Signaling Proteins Recruited to the Sperm Binding Site: Role of β-Catenin and Rho A

doi: 10.3389/fcell.2022.886664

Figure Lengend Snippet: Localization of β-catenin at the sperm binding site.Oocytes fixed at different stages of fertilization were labeled with anti- β-catenin (green) and Alexa Fluor-568 phalloidin (red) as well as Hoechst 33342 (blue). (A) Recently bound sperm. (B) bound sperm with microvilli exhibiting β-catenin labelling. (C) β-catenin plaque colocalizing with the actin layer. (D) sperm undergoing incorporation into the ooplasm with a single bright patch of β-catenin remaining. Images were selected from 30 oocytes recovered from 4 females. Magnification is indicated by the bar.

Article Snippet: Primary antibodies used include rabbit anti-human non-phospho (Active) β−catenin ((Ser33/37/Thr41, Cat # 8814), Cell Signaling, Danvers, MA), rabbit anti- β−catenin (epitope different from the non-phosphorylated antibody from Cell Signaling) [(Cat # BD610153) Becton, Dickinson, and Co. Franklin Lakes, NJ], rabbit anti-human RHOA (Cat. # 10749-1-AP, Proteintech, Rosemont, IL).

Techniques: Binding Assay, Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Signaling Proteins Recruited to the Sperm Binding Site: Role of β-Catenin and Rho A

doi: 10.3389/fcell.2022.886664

Figure Lengend Snippet: Distribution of f-actin and β-catenin in the oocyte cortex.MII oocyte from a virgin female was treated by prolonged fixation and labeled with anti- β-catenin (green) and Alexa Fluor-568 phalloidin (red) to label f-actin, as well as Hoechst 33342 (blue). Z-stack images viewed in volume mode (A) , a tangential image (B) , and an equatorial image (C) reveal fenestrae appearing as holes in the cortical actin layer (A,B) or as regions of thinner f-actin in the equatorial view (C) . Images were selected from 10 oocytes from three females. Magnification is indicated by the bar.

Article Snippet: Primary antibodies used include rabbit anti-human non-phospho (Active) β−catenin ((Ser33/37/Thr41, Cat # 8814), Cell Signaling, Danvers, MA), rabbit anti- β−catenin (epitope different from the non-phosphorylated antibody from Cell Signaling) [(Cat # BD610153) Becton, Dickinson, and Co. Franklin Lakes, NJ], rabbit anti-human RHOA (Cat. # 10749-1-AP, Proteintech, Rosemont, IL).

Techniques: Labeling

Journal: Journal of Cellular and Molecular Medicine

Article Title: Huoxin pill prevents acute myocardial ischaemia injury via inhibition of Wnt/β‑catenin signaling

doi: 10.1111/jcmm.17028

Figure Lengend Snippet: HXP is a potent inhibitor of Wnt/β‑catenin activation‐induced cardiac injury and DNA damage. (A‐C) Reporter gene assay using TOPflash analysis. Wnt3a (A), LiCl addition (B), or full‐length LRP6 (C) were transfected with TOPflash in HEK293 cells for 48 h. HXP or LiCl were directly added to the transfected cells at varying concentrations, as indicated. RLU, relative light units. Values are presented as the mean ± sem. n.s., no significance, * p < 0.05, *** p < 0.001 versus control group. n = 3 for each experiment. (D) Representative immunoblot (left) and quantification (right) showing expressions of γ‐H2AX and β‐catenin following siRNA‐mediated knockdown of β‐catenin for 48 hours, subsequent pretreatment with 1 µg ml −1 HXP for 2 hours and treatment with 100 µM H 2 O 2 for 30 minutes. GAPDH was used as the loading control. Data are presented as mean ± sem, * p < 0.05, ** p < 0.01. n = 3 for each. (E, F) Representative immunoblot showing the expression of cytoplasmic phosphorylated‐β‐catenin (E) and nuclear β‐catenin (F) in the infarct region of mice treated with PBS, low‐dose HXP‐L (3 mg kg d −1 ), or high‐dose HXP‐H (9 mg kg d −1 ) at 1 h and 2 h post‐MI. GAPDH was used as the cytoplasmic loading control. TBP was used as the nuclear loading control

Article Snippet: Collagen‐I (14695–1‐AP), Collagen‐III (22734–1‐AP), ACTA‐1 (17521–1‐AP), ACTN2 (14221–1‐AP), GAPDH (60004–1‐Ig), TBP (22006–1‐AP) were purchased from Proteintech. β‐catenin (8480S), Phospho‐β‐Catenin (p‐β‐catenin; 9561S) were provided by CST.

Techniques: Activation Assay, Reporter Gene Assay, Transfection, Control, Western Blot, Knockdown, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Huoxin pill prevents acute myocardial ischaemia injury via inhibition of Wnt/β‑catenin signaling

doi: 10.1111/jcmm.17028

Figure Lengend Snippet: HXP attenuates H 2 O 2 ‐induced DNA damage via inhibition of Wnt/β‐catenin pathway. (A) Representative immunoblot (top) and quantification (bottom) showing the expression of nuclear β‐catenin following LiCl stimulation at different time intervals in AC16 cardiomyocytes. TBP was used as the loading control. n.s., no significance, * p < 0.05, ** p < 0.01 versus control group. n = 3 for each experiment. (B) Representative immunoblot (top) and quantification (bottom) showing the expression of nuclear β‐catenin following LiCl stimulation at different concentrations in AC16 cardiomyocytes. TBP was used as the loading control. n.s., no significance, * p < 0.05, ** p < 0.01 versus control group. n = 3 for each experiment. (C) Representative immunoblot (left) and quantification (right) showing the expression of γ‐H2AX and β‐catenin following pretreatment with 1 µg ml −1 HXP for 2 hours, 40 mM LiCl for 4 hours, and subsequent treatment with 100 µM H 2 O 2 for 30 minutes. GAPDH was used as the loading control. Data are presented as mean ± sem, * p < 0.05. n = 3 for each

Article Snippet: Collagen‐I (14695–1‐AP), Collagen‐III (22734–1‐AP), ACTA‐1 (17521–1‐AP), ACTN2 (14221–1‐AP), GAPDH (60004–1‐Ig), TBP (22006–1‐AP) were purchased from Proteintech. β‐catenin (8480S), Phospho‐β‐Catenin (p‐β‐catenin; 9561S) were provided by CST.

Techniques: Inhibition, Western Blot, Expressing, Control

Journal: Frontiers in cell and developmental biology

Article Title: USP36-Mediated Deubiquitination of DOCK4 Contributes to the Diabetic Renal Tubular Epithelial Cell Injury via Wnt/β-Catenin Signaling Pathway.

doi: 10.3389/fcell.2021.638477

Figure Lengend Snippet: FIGURE 4 | USP36 regulates the ubiquitination level of DOCK4. (A) String website was performed to predict that USP36 can bind to DOCK4. (B) Immunoprecipitation analysis was performed with cell lysates of TECs with anti-USP36 or anti-DOCK4 antibodies, n = 3. (C) Interaction of USP36 and DOCK4 in TECs visualized by Duolink proximity ligation assay, n = 3. (D) RT-qPCR analysis of the mRNA level of DOCK4 after knockdown or overexpression of USP36. Unpaired, two-tailed t tests were used to compare groups, n = 3, ns, p > 0.05. (E,F) WB analysis of the protein levels of USP36 and DOCK4 in TECs after knockdown or overexpression of USP36. Data are presented as the mean ± S.D, n = 3, *p < 0.05, **p < 0.01. (G,H) WB analysis of the protein levels of USP36 and the ub-DOCK4 in TECs after knockdown or overexpression of USP36. Data are presented as the mean ± S.D, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001. (I) Ub-DOCK4 was detected by Duolink proximity ligation assay after knockdown or overexpression of USP36. Scale bars = 25 µm, n = 3. (J) Duolink proximity ligation assay analysis of ub-DOCK4 expression after knockdown or overexpression of USP36. Data are presented as the mean ± S.D, n = 3, **p < 0.01, ***p < 0.001.

Article Snippet: Primary antibodies were used as follows: rabbit antiUSP36 antibody (14783-1-AP, Proteintech,1:1,000), rabbit antiDOCK4 antibody (21861-1-AP, Proteintech,1:1,000), rabbit antiβ-catenin antibody (51067-1-AP, Proteintech, 1:5,000), rabbit anti-E-cad antibody (20874-1-AP, Proteintech,1:5,000), rabbit anti-α-SMA antibody (55135-1-AP, Proteintech,1:2,000).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Proximity Ligation Assay, Quantitative RT-PCR, Knockdown, Over Expression, Two Tailed Test, Expressing

Journal: Frontiers in cell and developmental biology

Article Title: USP36-Mediated Deubiquitination of DOCK4 Contributes to the Diabetic Renal Tubular Epithelial Cell Injury via Wnt/β-Catenin Signaling Pathway.

doi: 10.3389/fcell.2021.638477

Figure Lengend Snippet: FIGURE 5 | USP36 regulates EMT induced by high glucose of TECs via DOCK4-dependent Wnt/β-catenin signaling pathway. (A,B) Immunohistochemistry staining for DOCK4 expression in human kidney sections. Scale bars = 100 µm, n = 3. (C,D) Immunohistochemistry staining for USP36 expression in mouse renal tissues. Scale bars = 100 µm, n = 3. (E,F) WB analysis of the protein levels of DOCK4 in high glucose induced TECs. Data are presented as the mean ± S.D, n = 3, *p < 0.05. (G,H) WB analysis of the protein levels of DOCK4, β-catenin, α-SMA and E-cad in TECs after knockdown of DOCK4. Data are presented as the mean ± S.D, n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001. (I,J) WB analysis of the protein levels of USP36, β-catenin, α-SMA and E-cad in TECs after knockdown of USP36. Data are presented as the mean ± S.D, n = 3, *p < 0.05, and **p < 0.01. (K,L) WB analysis of the protein levels of USP36, β-catenin, α-SMA and E-cad in TECs exposed to long time high glucose following the knockdown of USP36. Data are presented as the mean ± S.D, n = 3, *p < 0.05, and **p < 0.01. (M,N) WB analysis of the protein levels of USP36, DOCK4, β-catenin, α-SMA, and E-cad after co-transfected with USP36-overexpressing and DOCK4-silencing siRNA. Data are presented as the mean ± S.D, n = 3, *p < 0.05, and **p < 0.01.

Article Snippet: Primary antibodies were used as follows: rabbit antiUSP36 antibody (14783-1-AP, Proteintech,1:1,000), rabbit antiDOCK4 antibody (21861-1-AP, Proteintech,1:1,000), rabbit antiβ-catenin antibody (51067-1-AP, Proteintech, 1:5,000), rabbit anti-E-cad antibody (20874-1-AP, Proteintech,1:5,000), rabbit anti-α-SMA antibody (55135-1-AP, Proteintech,1:2,000).

Techniques: Immunohistochemistry, Staining, Expressing, Knockdown, Transfection

Journal: American Journal of Translational Research

Article Title: RNF128 suppresses the malignancy of colorectal cancer cells via inhibition of Wnt/β-catenin signaling

doi:

Figure Lengend Snippet: Loss of RNF128 increases β-catenin protein level and its target genes in colorectal cancer. (A) The mRNA expressions of cyclin D1, cyclin E1, cyclin A1 and cyclin B1 were quantified in RNF128 knockdown or overexpression cells compared to their respective controls in DLD1. (B) β-catenin, CCND1, and RNF128 expressions were confirmed using Western blotting. β-actin was used as a loading control. (C) Expression of β-catenin and RNF128 in nuclear and cytoplasmic fractions in RNF128-knockdown cells and control cells of DLD1. β-tubulin was used as the cytoplasmic reference protein. LaminB1 was used as the nuclear reference protein. *P<0.05, **P<0.01 and ***P<0.001. No significant difference (P>0.05, ns). Data represented as mean ± SD.

Article Snippet: Supernatants were collected for measuring protein concentration by BCA assay (Thermofisher Scientific, USA). β-tubulin Rabbit mAb, Lamin B1 Rabbit mAb and β-actin Rabbit mAb were purchased from Proteintech (china); β-Catenin Rabbit mAb and CCND1 Rabbit mAb were from Cell Signaling Technology (USA); Ub and RNF128 Rat mAb were from Santa Cruz Biotechnology (USA); goat anti-rabbit and goat anti-rat were from Cell Signaling Technology (USA).

Techniques: Knockdown, Over Expression, Western Blot, Control, Expressing

Journal: American Journal of Translational Research

Article Title: RNF128 suppresses the malignancy of colorectal cancer cells via inhibition of Wnt/β-catenin signaling

doi:

Figure Lengend Snippet: RNF128 directly interacts with and ubiquitinates β-catenin for degradation. (A and B) co-immunoprecipitation (co-IP) was performed with anti-RNF128 antibody (A) or β-catenin antibody (B) and anti-lgG antibody in DLD1 cells. (C) DLD1 cells were treated with cycloheximide at the indicated time and β-catenin levels were examined by Western blotting. (D) MG132 (10 μM) was added to RNF128-overexpression cells and overexpression control cells of DLD1 for 6 h. The relative changes of β-catenin were analyzed. (E) Ubiquitination of β-catenin was analyzed by the co-IP assay and immunoblotting with anti-ubiquitin antibody in DLD1. (F) Model of the suppressive role of RNF128 in Wnt/β-catenin/CCND1 pathway. The binding of Wnt to Frizzled (FZD) receptors activates DVL. Phosphorylated DVL stabilizes cytoplasmic β-catenin to promote nuclear translocation of β-catenin. The entry of β-catenin into the nucleus interacts with TCF/LEF and enhances transcription of downstream target (CCND1). RNF128 increases β-catenin ubiquitination in this process. *P<0.05, **P<0.01 and ***P<0.001. No significant difference (P>0.05, ns). Data represented as mean ± SD.

Article Snippet: Supernatants were collected for measuring protein concentration by BCA assay (Thermofisher Scientific, USA). β-tubulin Rabbit mAb, Lamin B1 Rabbit mAb and β-actin Rabbit mAb were purchased from Proteintech (china); β-Catenin Rabbit mAb and CCND1 Rabbit mAb were from Cell Signaling Technology (USA); Ub and RNF128 Rat mAb were from Santa Cruz Biotechnology (USA); goat anti-rabbit and goat anti-rat were from Cell Signaling Technology (USA).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Over Expression, Control, Ubiquitin Proteomics, Binding Assay, Translocation Assay

Journal: American Journal of Translational Research

Article Title: RNF128 suppresses the malignancy of colorectal cancer cells via inhibition of Wnt/β-catenin signaling

doi:

Figure Lengend Snippet: RNF128 inhibits colorectal cancer cell proliferation in vivo. (A) Construction and evaluation of implanted model of SW480-sh-RNF128 cells or their control cells in nude nice (n=4). (B and C) The volumes and weights of tumor. (D) β-catenin, RNF128 expression and the ubiquitin level of b-catenin in the tumorigenic tissue of the mouse were confirmed using Western blotting. (E) RNF128 expression in the tumorigenic tissues of the mouse was determined by qRT-PCR. **P<0.01. No significant difference (P>0.05, ns). Data represented as mean ± SD.

Article Snippet: Supernatants were collected for measuring protein concentration by BCA assay (Thermofisher Scientific, USA). β-tubulin Rabbit mAb, Lamin B1 Rabbit mAb and β-actin Rabbit mAb were purchased from Proteintech (china); β-Catenin Rabbit mAb and CCND1 Rabbit mAb were from Cell Signaling Technology (USA); Ub and RNF128 Rat mAb were from Santa Cruz Biotechnology (USA); goat anti-rabbit and goat anti-rat were from Cell Signaling Technology (USA).

Techniques: In Vivo, Control, Expressing, Ubiquitin Proteomics, Western Blot, Quantitative RT-PCR

Journal: Experimental and Therapeutic Medicine

Article Title: Psoralen, a natural phytoestrogen, improves diaphyseal fracture healing in ovariectomized mice: A preliminary study

doi: 10.3892/etm.2021.9799

Figure Lengend Snippet: Expression of estrogen receptor. (A-F) Immunohistochemistry showing ER-α and ER-β staining in the fracture callus of OVX and psoralen+OVX groups at 21 days post fracture. Magnification, x40. (G-J) Western blot analyses were performed to determine the expression levels of ER-α and ER-β in OVX and psoralen+OVX groups. Data are presented as the mean ± SD. * P<0.05 vs. OVX group. OVX, ovariectomy; ER, estrogen receptor.

Article Snippet: Consequently, the membranes were incubated at 4˚C overnight with anti-mouse ER-α (cat. no. MS-354-P0; Thermo Fisher Scientific, Inc.; 1:1,000), rabbit anti-mouse ER-β (cat. no. 14007-1-AP; ProteinTech Group, Inc.; 1:1,000) and anti-mouse β-actin (cat. no. GB12001; Wuhan Servicebio Technology Co., Ltd.; 1:3,000) antibodies, and subsequently with certain horseradish peroxidase-conjugated secondary antibodies, including ER-α (cat. no. GB11205; Wuhan Servicebio Technology Co., Ltd. 1:3,000), β-actin (cat. no. GB12001, Wuhan Servicebio Technology Co., Ltd. 1:3,000) and ER-β (cat. no. 14007-1-AP; Wuhan Sanying Biotechnology.

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot

Journal: iScience

Article Title: Neuroepithelial cell-transforming 1 promotes cardiac fibrosis via the Wnt/β-catenin signaling pathway

doi: 10.1016/j.isci.2023.107888

Figure Lengend Snippet: Effect of NET1 knockdown on TGF-β1-induced cardiac fibroblasts (A) mRNA change of NET1, n = 4, ∗p = 0.0286 TGF-β1 vs. Ctrl by Mann Whitney test. ∗∗p = 0.0084 TGF-β1 + si-NET1 vs. TGF-β1，∗p = 0.0360 vs. TGF-β1 + si-NET1 vs. TGF-β1 + NC by Kruskal-Wallis test. (B) mRNA of collagen I (COL1A1) was detected by qRT-PCR, n = 4, ∗p = 0.0286 TGF-β1 vs. Ctrl by Mann Whitney test. ∗p = 0.0360 TGF-β1 + si-NET1 vs. TGF-β1，∗p = 0.0140 TGF-β1 + si-NET1 vs. TGF-β1 + NC by Kruskal-Wallis test. (C) Representative image of cardiac fibroblasts stained with α-SMA antibody (Red) and NET1 antibody (Green). Scale bar: 50 μm, magnification 200×. (D) The migration of cardiac fibroblast was detected by scratch assay, n = 5, ∗p = 0.0452 TGF-β1 vs. Ctrl, ∗p = 0.0277 TGF-β1 + si-NET1 vs. TGF-β1, ∗p = 0.0452 TGF-β1 + si-NET1 vs. TGF-β1 + NC by Kruskal-Wallis test. (E) The proliferative ability of cardiac fibroblasts was detected by EdU kit. Scale bar: 50 μm, magnification 200×, n = 4, ∗p = 0.0313 TGF-β1 vs. Ctrl, ∗∗p = 0.0075 TGF-β1 + si-NET1 vs. TGF-β1, ∗p = 0.0143 TGF-β1 + si-NET1 vs. TGF-β1 + NC by Kruskal-Wallis test. (F) The viability of cardiac fibroblasts was detected by MTT assay, n = 9, ∗p = 0.0465 TGF-β1 vs. Ctrl, ∗∗p < 0.0001 TGF-β1 + si-NET1 vs. TGF-β1, ∗∗p < 0.0001 TGF-β1 + si-NET1 vs. TGF-β1 + NC by Kruskal-Wallis test. For all statistical plots, the data are presented as mean ± SEM.

Article Snippet: The protein was first cultured with 5% non-fat dry milk for 1.5 hours, followed by an overnight incubation at 4°C with primary antibodies against NET1 (Proteintech, 28180-1-AP), alpha-smooth muscle actin (α-SMA; Proteintech, 55135-1-AP), fibronectin 1 (FN-1, Proteintech, USA), collagen I (Wanleibio, WL0088), β-catenin (Proteintech, 51067-2-AP), GAPDH (Proteintech, 60004-1-Ig), α-actinin (Proteintech, USA), glycogen synthase kinase-3β (GSK3β; Proteintech, 22104-I-AP), GSK3β- serine 9 (S9) (Proteintech, 67588-7-Ig), and GSK3β- serine 389 (S389) (Proteintech, 14850-I-AP).

Techniques: MANN-WHITNEY, Quantitative RT-PCR, Staining, Migration, Wound Healing Assay, MTT Assay

Journal: iScience

Article Title: Neuroepithelial cell-transforming 1 promotes cardiac fibrosis via the Wnt/β-catenin signaling pathway

doi: 10.1016/j.isci.2023.107888

Figure Lengend Snippet: Effect of NET1 overexpression on cardiac fibroblasts (A) Western blot was used to detect the protein expression levels of NET1, FN-1, and collagen I in cardiac fibroblasts transfected with NET1 overexpression plasmid, n = 4, ∗p = 0.0286 NET1 vs. Vector by Mann Whitney test. qRT-PCR was used to detect NET1, COL1A1 in fibroblasts and COL3A3 mRNA expression levels. n = 4–5, ∗p = 0.0286, ∗p = 0.0286, ∗∗p = 0.0079 NET1 vs. Vector by Mann Whitney test. (B) Fibroblast viability was detected by MTT assay, n = 9, ∗p = 0.0244 NET1 vs. Vector by Mann Whitney test. (C) The expression of myofibroblast marker α-SMA was detected by immunofluorescence (n = 5, ∗∗p = 0.0079) and western blot (n = 8, ∗∗p = 0.0019). NET1 vs. Vector by Mann Whitney test, Scale bar: 50 μm, magnification 200×. (D) Fibroblast migration was detected by cell scratch assay. n = 4, ∗p = 0.0286 NET1 vs. Vector by Mann Whitney test. (E) Fibroblast proliferation detected by Edu kit. Scale bar: 50 μm, magnification 200×, n = 3, p = 0.1000 NET1 vs. Vector by Mann Whitney test. (F) Western blot was used to detect the protein level of β-catenin. n = 4, ∗p = 0.0286 NET1 vs. Vector by Mann Whitney test. For all statistical plots, the data are presented as mean ± SEM. (G) Confocal microscopy was used to detect the expression of β-catenin in the nuclei of fibroblasts transfected with NET1 overexpression plasmid and Vector. Scale bar: 10 μm.

Article Snippet: The protein was first cultured with 5% non-fat dry milk for 1.5 hours, followed by an overnight incubation at 4°C with primary antibodies against NET1 (Proteintech, 28180-1-AP), alpha-smooth muscle actin (α-SMA; Proteintech, 55135-1-AP), fibronectin 1 (FN-1, Proteintech, USA), collagen I (Wanleibio, WL0088), β-catenin (Proteintech, 51067-2-AP), GAPDH (Proteintech, 60004-1-Ig), α-actinin (Proteintech, USA), glycogen synthase kinase-3β (GSK3β; Proteintech, 22104-I-AP), GSK3β- serine 9 (S9) (Proteintech, 67588-7-Ig), and GSK3β- serine 389 (S389) (Proteintech, 14850-I-AP).

Techniques: Over Expression, Western Blot, Expressing, Transfection, Plasmid Preparation, MANN-WHITNEY, Quantitative RT-PCR, MTT Assay, Marker, Immunofluorescence, Migration, Wound Healing Assay, Confocal Microscopy

Journal: iScience

Article Title: Neuroepithelial cell-transforming 1 promotes cardiac fibrosis via the Wnt/β-catenin signaling pathway

doi: 10.1016/j.isci.2023.107888

Figure Lengend Snippet: NET1 may be involved in cardiac fibrosis through β-catenin (A) Cardiac fibroblasts were transfected with si-β-catenin and NC, and the protein level of β-catenin was detected by western blot. n = 5,∗p = 0.0175 si-β-catenin1 vs. NC, ∗p = 0.0327 si-β-catenin2 vs. NC by Kruskal-Wallis test. (B and C) Fibrosis marker protein FN-1 (n = 4, ∗∗p = 0.0011 NET1 + si-β-catenin vs. NET1 by Kruskal-Wallis test) and (C) collagen I expression was detected by western blot. n = 4, ∗∗p = 0.0075 NET1 vs. Ctrl, ∗p = 0.0259 NET1 + si-β-catenin vs. NET1 by Kruskal-Wallis test. (D) MTT method was used for detection of fibroblast viability. n = 7, ∗∗p = 0.0093 Ctrl vs. NET1, ∗∗p = 0.0063 NET1 + si-β-catenin vs. NET1 by Kruskal-Wallis test. (E) Fibroblast migration ability was detected by cell scratch method. n = 4, ∗p = 0.0166 Ctrl vs. NET1, ∗∗p = 0.0003 NET1 + si-β-catenin vs. NET1 by Kruskal-Wallis test. For all statistical plots, the data are presented as mean ± SEM. (F) Co-immunoprecipitation (Co-IP) was performed to verify GSK3β protein interaction with NET1 and β-catenin.

Article Snippet: The protein was first cultured with 5% non-fat dry milk for 1.5 hours, followed by an overnight incubation at 4°C with primary antibodies against NET1 (Proteintech, 28180-1-AP), alpha-smooth muscle actin (α-SMA; Proteintech, 55135-1-AP), fibronectin 1 (FN-1, Proteintech, USA), collagen I (Wanleibio, WL0088), β-catenin (Proteintech, 51067-2-AP), GAPDH (Proteintech, 60004-1-Ig), α-actinin (Proteintech, USA), glycogen synthase kinase-3β (GSK3β; Proteintech, 22104-I-AP), GSK3β- serine 9 (S9) (Proteintech, 67588-7-Ig), and GSK3β- serine 389 (S389) (Proteintech, 14850-I-AP).

Techniques: Transfection, Western Blot, Marker, Expressing, Migration, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: iScience

Article Title: Neuroepithelial cell-transforming 1 promotes cardiac fibrosis via the Wnt/β-catenin signaling pathway

doi: 10.1016/j.isci.2023.107888

Figure Lengend Snippet: Effects of GSK3β knockdown on TGF-β1-induced cardiac fibrosis (A and B) Expression level of NET1 (n = 3, ∗∗p = 0.0092 TGF-β1 vs. Ctrl, ∗p = 0.0127 TGF-β1 + siNC vs. Ctrl by Kruskal-Wallis test) (B) collagen I (n = 3, ∗p = 0.0415 TGF-β1 vs. Ctrl, ∗p = 0.0315 TGF-β1 + siNC vs. Ctrl, ∗p = 0.0415 TGF-β1 + siGSK3β vs. TGF-β1 + siNC) and hydroxyproline (n = 4, ∗∗p = 0.0014 TGF-β1 vs. Ctrl, ∗p = 0.0213 TGF-β1 + siNC vs. Ctrl, ∗p = 0.0143 TGF-β1 + siGSK3β vs. TGF-β1 by Kruskal-Wallis test. (C) α-SMA after knockdown GSK3β in cardiac fibroblasts. Scale bar: 20 μm, magnification, 200×. (D) EdU staining was used to detect fibroblast proliferation. Scale bar: 20 μm, magnification, 200×. n = 6, ∗∗p = 0.0005 TGF-β1 vs. Ctrl, ∗∗p = 0.0004 TGF-β1 + siNC vs. Ctrl, ∗p = 0.0160 TGF-β1 + siGSK3β vs. TGF-β1 + siNC by Kruskal-Wallis test. (E) The migration of fibroblasts was detected by scratch experiment. n = 3, ∗∗p = 0.0046, TGF-β1 + siGSK3β vs. TGF-β1, ∗p = 0.0235, TGF-β1 + siGSK3β vs. TGF-β1 + siNC by Kruskal-Wallis test. (F) Expression level of GSK3β after knockdown GSK3β in cardiac fibroblasts. n = 3, ∗∗p = 0.0055, TGF-β1 + siGSK3β vs. TGF-β1, ∗p = 0.0201, TGF-β1 + siGSK3β vs. TGF-β1 + siNC by Kruskal-Wallis test. (G and H) Expression level of GSK3β-S389, and (H) GSK3β-S9 after knockdown GSK3β in cardiac fibroblasts. Scale bar: 20 μm, magnification, 200×. (I) Confocal microscopy was used to detect the expression of β-catenin in the nuclei of fibroblasts transfected with TGF-β1 and knockdown GSK3β. Expression level of β-catenin after knockdown GSK3β in cardiac fibroblasts. n = 3, ∗p = 0.0415 TGF-β1 vs. Ctrl, ∗p = 0.0174 TGF-β1 + siNC vs. Ctrl, ∗p = 0.0415 TGF-β1 + siGSK3β vs. TGF-β1 + siNC by Kruskal-Wallis test. For all statistical plots, the data are presented as mean ± SEM.

Article Snippet: The protein was first cultured with 5% non-fat dry milk for 1.5 hours, followed by an overnight incubation at 4°C with primary antibodies against NET1 (Proteintech, 28180-1-AP), alpha-smooth muscle actin (α-SMA; Proteintech, 55135-1-AP), fibronectin 1 (FN-1, Proteintech, USA), collagen I (Wanleibio, WL0088), β-catenin (Proteintech, 51067-2-AP), GAPDH (Proteintech, 60004-1-Ig), α-actinin (Proteintech, USA), glycogen synthase kinase-3β (GSK3β; Proteintech, 22104-I-AP), GSK3β- serine 9 (S9) (Proteintech, 67588-7-Ig), and GSK3β- serine 389 (S389) (Proteintech, 14850-I-AP).

Techniques: Expressing, Staining, Migration, Confocal Microscopy, Transfection

Journal: iScience

Article Title: Neuroepithelial cell-transforming 1 promotes cardiac fibrosis via the Wnt/β-catenin signaling pathway

doi: 10.1016/j.isci.2023.107888

Figure Lengend Snippet: Effects of GSK3β knockdown on cardiac fibrosis induced by NET1 overexpression (A) Expression level of NET1 after overexpression NET1 and knockdown GSK3β in cardiac fibroblasts. n = 3, ∗∗p = 0.0022 NET1 vs. NC + siGSK3β, n = 3, by Kruskal-Wallis test. (B) Expression level of collagen I (n = 3, ∗∗p = 0.0022 NET1 vs. NC + siGSK3β) and hydroxyproline (n = 3, ∗p = 0.0415 NC vs. NET1, ∗∗p = 0.0022 NET1 vs. NC + siGSK3β, ∗p = 0.0415 NET1+ siGSK3β vs. NC + siGSK3β) after overexpression NET1 and knockdown GSK3β in cardiac fibroblasts. (C) Expression level of α-SMA after overexpression NET1 and knockdown GSK3β in cardiac fibroblasts. Scale bar: 20 μm, magnification, 200×. (D) EdU staining was used to detect fibroblast proliferation, Scale bar: 20 μm, magnification, 200×. n = 3. ∗p = 0.0415 NC vs. NET1, ∗∗p = 0.0022 siGSK3β vs. NET1, ∗p = 0.0415 NET1 + siGSK3β vs. siGSK3β by Kruskal-Wallis test. (E) The migration of fibroblasts was detected by scratch experiment. n = 3, ∗p = 0.0134 siGSK3β vs. NET1 by Kruskal-Wallis test. (F) Expression level of β-catenin after overexpression NET1 and knockdown GSK3β in cardiac fibroblasts. n = 3, ∗p = 0.0280 NC vs. NET1, ∗∗p = 0.0029 NET1 vs. NC + siGSK3β, by Kruskal-Wallis test. (G) Expression level of GSK3β after overexpression NET1 and knockdown GSK3β in cardiac fibroblasts. n = 3, ∗p = 0.0174 siGSK3β vs. NET1, ∗∗p = 0.0066 NET1 + siGSK3β vs. NET1 by Kruskal-Wallis test. For all statistical plots, the data are presented as mean ± SEM. (H and I) Expression level of GSK3β-S389 (Green), GSK3β-S9 (Red) (I), and β-catenin after overexpression NET1 and knockdown GSK3β in cardiac fibroblasts. Scale bar: 20 μm, magnification, 200×.

Article Snippet: The protein was first cultured with 5% non-fat dry milk for 1.5 hours, followed by an overnight incubation at 4°C with primary antibodies against NET1 (Proteintech, 28180-1-AP), alpha-smooth muscle actin (α-SMA; Proteintech, 55135-1-AP), fibronectin 1 (FN-1, Proteintech, USA), collagen I (Wanleibio, WL0088), β-catenin (Proteintech, 51067-2-AP), GAPDH (Proteintech, 60004-1-Ig), α-actinin (Proteintech, USA), glycogen synthase kinase-3β (GSK3β; Proteintech, 22104-I-AP), GSK3β- serine 9 (S9) (Proteintech, 67588-7-Ig), and GSK3β- serine 389 (S389) (Proteintech, 14850-I-AP).

Techniques: Over Expression, Expressing, Staining, Migration

Journal: iScience

Article Title: Neuroepithelial cell-transforming 1 promotes cardiac fibrosis via the Wnt/β-catenin signaling pathway

doi: 10.1016/j.isci.2023.107888

Figure Lengend Snippet: The role of NET1 in myocardial infarction in mice (A) The protein level of α-SMA in mouse model. n = 5, ∗∗p = 0.0088 Sham vs. MI, ∗∗p < 0.0001 MI vs. KO, ∗∗p = 0.0088 KO vs. KO + MI, by Kruskal-Wallis test, ∗∗p = 0.0079 KO + MI vs. MI by Mann Whitney test. (B) The protein level of collagen I (n = 3, ∗p = 0.0415 Sham vs. MI, ∗∗p = 0.0022 MI vs. KO, ∗p = 0.0415 KO vs. KO + MI) and hydroxyproline in mouse model (n = 6, ∗∗p = 0.0013 Sham vs. MI, ∗∗p = 0.0046 MI vs. KO, ∗p = 0.0479 MI vs. KO + MI by Kruskal-Wallis test). (C and D) The protein level of NET1 in mouse model. n = 3, ∗∗p = 0.0032 MI vs. KO, ∗p = 0.0415 MI vs. KO + MI. (D) The protein level of β-catenin in mouse MI model. n = 4, ∗p = 0.0143 Sham vs. MI, ∗∗p = 0.0005 MI vs. KO, ∗p = 0.0213 KO vs. KO + MI by Kruskal-Wallis test. ∗p = 0.0286 KO + MI vs. MI by Mann Whitney test. (E) The protein level of GSK3β-S389 in mouse MI model. n = 3, ∗p = 0.0022 MI vs. KO by Kruskal-Wallis test. The protein level of GSK3β-S9 in mouse MI model. n = 3∗p = 0.0022 MI vs. KO by Kruskal-Wallis test. For all statistical plots, the data are presented as mean ± SEM. (F) Changes of myocardial ultrastructure in each group. The ultrastructure of cardiomyocytes was observed under electron microscope. Magnification, ×15000. Scale bar calibration: 2 μm; The heart tissues of mice in each group were observed by Masson staining. Masson staining was used to observe the heart tissue. Scale bar calibration: 100 μm.

Article Snippet: The protein was first cultured with 5% non-fat dry milk for 1.5 hours, followed by an overnight incubation at 4°C with primary antibodies against NET1 (Proteintech, 28180-1-AP), alpha-smooth muscle actin (α-SMA; Proteintech, 55135-1-AP), fibronectin 1 (FN-1, Proteintech, USA), collagen I (Wanleibio, WL0088), β-catenin (Proteintech, 51067-2-AP), GAPDH (Proteintech, 60004-1-Ig), α-actinin (Proteintech, USA), glycogen synthase kinase-3β (GSK3β; Proteintech, 22104-I-AP), GSK3β- serine 9 (S9) (Proteintech, 67588-7-Ig), and GSK3β- serine 389 (S389) (Proteintech, 14850-I-AP).

Techniques: MANN-WHITNEY, Microscopy, Staining

Journal: iScience

Article Title: Neuroepithelial cell-transforming 1 promotes cardiac fibrosis via the Wnt/β-catenin signaling pathway

doi: 10.1016/j.isci.2023.107888

Figure Lengend Snippet:

Article Snippet: The protein was first cultured with 5% non-fat dry milk for 1.5 hours, followed by an overnight incubation at 4°C with primary antibodies against NET1 (Proteintech, 28180-1-AP), alpha-smooth muscle actin (α-SMA; Proteintech, 55135-1-AP), fibronectin 1 (FN-1, Proteintech, USA), collagen I (Wanleibio, WL0088), β-catenin (Proteintech, 51067-2-AP), GAPDH (Proteintech, 60004-1-Ig), α-actinin (Proteintech, USA), glycogen synthase kinase-3β (GSK3β; Proteintech, 22104-I-AP), GSK3β- serine 9 (S9) (Proteintech, 67588-7-Ig), and GSK3β- serine 389 (S389) (Proteintech, 14850-I-AP).

Techniques: Recombinant, Saline, In Vitro, Lysis, CRISPR, Knock-Out, Software