Journal: PLoS ONE

Article Title: The molecular connection of histopathological heterogeneity in hepatocellular carcinoma: A role of Wnt and Hedgehog signaling pathways

doi: 10.1371/journal.pone.0208194

Figure Lengend Snippet: Primer set of various genes adopted in RT-PCR.

Article Snippet: Primary antibodies β-Catenin (Cat: SC-7963), Wnt3 (Cat: ab50341), PCNA (Cat: SC-56), Gli1 (Cat: SC-20687), Gli2 (Cat: SC-28674), sFRP1 (Cat: ab126613), MMP9 (Cat: SC-393859), GSK3β (Cat: SC-9166), Shh (Cat: SC-9024), CyclinD1(Cat:SC-20044), CyclinB1(Cat:SC-752) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques:

Journal: PLoS ONE

Article Title: The molecular connection of histopathological heterogeneity in hepatocellular carcinoma: A role of Wnt and Hedgehog signaling pathways

doi: 10.1371/journal.pone.0208194

Figure Lengend Snippet: Enhanced Wnt and Hh signaling correlates with moderately differentiated HCC. (A) H and E stained liver sections of control and promotion group (group2) animals showing moderately differentiated, thick trabecular pattern, followed by photomicraphs showing IHC staining of Wnt and Hh pathway molecules at TE and TL phases of promotion stage. The corresponding IHC staining intensity graph is shown in the side panel of figures.Y-axis represents labeling index (%) visual score in each intensity graph. (B) Increasing mRNA expression level of Wnt and Hh pathway molecules of control and group2 animals at TE and TL phases of promotion stage. (C) The figure represents fold increase in relative mRNA expression, calculated with respect to GAPDH for Shh, Ptch1, Gli1, Wnt3 β-catenin in control and group2 animals at TE and TL phases of promotion stage (D) Analysis of Wnt and Hh pathway molecules in rat tissue lysates at TE and TL phases of promotion stage by ELISA. Data presented are representative of three independent experiments performed in triplicates and expressed as Mean±S.D. ** and *** differs significantly at p <0.005 and <0.0005.

Article Snippet: Primary antibodies β-Catenin (Cat: SC-7963), Wnt3 (Cat: ab50341), PCNA (Cat: SC-56), Gli1 (Cat: SC-20687), Gli2 (Cat: SC-28674), sFRP1 (Cat: ab126613), MMP9 (Cat: SC-393859), GSK3β (Cat: SC-9166), Shh (Cat: SC-9024), CyclinD1(Cat:SC-20044), CyclinB1(Cat:SC-752) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Staining, Control, Immunohistochemistry, Labeling, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Clinical science (London, England : 1979)

Article Title: Restoration of the gut barrier integrity and restructuring of the gut microbiome in aging by angiotensin-(1–7)

doi: 10.1042/CS20220904

Figure Lengend Snippet: List of antibodies used for western blotting and immunohistochemistry

Article Snippet: Antibody Catalog number Vendor Concentration Claudin 1 (Host - Ms) 37-4900 Thermofisher 1:100 Occludin (Host -Rb) 40-4700 Thermofisher 1:100 Lgr5 (Host – Ms) MA5-25644 Thermofisher 1:100 Olfm4 (Host – Rb) PA5-115687 Thermofisher 1:50 F4/80 (Host - Rat) 14-4801-82 Invitrogen 1:50 CD11b (Host - Rb) ab133357 Abcam 1:4000 633 Goat Anti Rb 20122 Biotium 1:250 633 Goat Anti Ms 20010 Biotium 1:250 488 Goat Anti Rat A11006 Invitrogen 1:100 DAPI 40043 Biotium - ACE MA5- 32741 Thermofisher 1: 1000 ACE-2 MA5- 32307 Thermofisher 1: 1000 MAS1 (G-1) Sc- 390453 Santacruz 1: 1000 AT-1 (G-3) Sc- 515884 Santacruz 1: 1000 AT-2 MA5- 32293 Thermofisher 1: 1000 Wnt3a TA801736S Origene 1:1000 (western blot) and 1:100 (IHC) Open in a separate window List of antibodies used for western blotting and immunohistochemistry.

Techniques: Western Blot, Concentration Assay

Journal: Clinical science (London, England : 1979)

Article Title: Restoration of the gut barrier integrity and restructuring of the gut microbiome in aging by angiotensin-(1–7)

doi: 10.1042/CS20220904

Figure Lengend Snippet: (A) Shown were representative fluorescence images of Wnt3a immunohistochemistry with nuclear counter stain DAPI in colon sections derived from young, old and Ang-(1–7)-treated mice. (B) The number of Wnt3a-positive cells/villus was lower in the sections of old colon (n = 28 villi/5 mice/group) compared with the young (****P<0.0001, n = 30 villi/5 mice/group), which was increased in the Ang-(1–7)-treated old group (***P<0.001, n = 25 villi/5 mice/group). (C) Shown were representative Western blots of Wnt3a in the lysates of colon tissue or ex vivo-cultured organoids from different treatment groups. (D) Wnt3a protein levels were lower in the colons of old compared with the young (*P<0.05, n = 5 mice/group), which was increased in the colons derived from Ang-(1–7)-treated old group (*P<0.05, n = 5 mice/group). In the ex vivo-cultured organoids, decreased Wnt3a levels in the organoids from old compared with that derived from young colons, which was increased by treatment with Ang-(1–7) (*P<0.05, n = 5 mice/group). Data sets were analyzed by Kruskall–Wallis test followed by Dunn’s test.

Article Snippet: Antibody Catalog number Vendor Concentration Claudin 1 (Host - Ms) 37-4900 Thermofisher 1:100 Occludin (Host -Rb) 40-4700 Thermofisher 1:100 Lgr5 (Host – Ms) MA5-25644 Thermofisher 1:100 Olfm4 (Host – Rb) PA5-115687 Thermofisher 1:50 F4/80 (Host - Rat) 14-4801-82 Invitrogen 1:50 CD11b (Host - Rb) ab133357 Abcam 1:4000 633 Goat Anti Rb 20122 Biotium 1:250 633 Goat Anti Ms 20010 Biotium 1:250 488 Goat Anti Rat A11006 Invitrogen 1:100 DAPI 40043 Biotium - ACE MA5- 32741 Thermofisher 1: 1000 ACE-2 MA5- 32307 Thermofisher 1: 1000 MAS1 (G-1) Sc- 390453 Santacruz 1: 1000 AT-1 (G-3) Sc- 515884 Santacruz 1: 1000 AT-2 MA5- 32293 Thermofisher 1: 1000 Wnt3a TA801736S Origene 1:1000 (western blot) and 1:100 (IHC) Open in a separate window List of antibodies used for western blotting and immunohistochemistry.

Techniques: Fluorescence, Immunohistochemistry, Staining, Derivative Assay, Western Blot, Ex Vivo, Cell Culture

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Role of Chrysophanol in Epithelial-Mesenchymal Transition in Oral Cancer Cell Lines via a Wnt-3-Dependent Pathway

doi: 10.1155/2020/8373715

Figure Lengend Snippet: The effect of chrysophanol on the expression of EMT markers, Wnt-3, pGSK3 β , and GSK3 β . (a) FaDu cells were treated with either the control (0 μ M) or the indicated concentration (30 μ M) of chrysophanol (Cho) for 24 h. Cells were then harvested and the proteins were separated by SDS-PAGE, followed by immunoblotting with the indicated antibodies. β -actin was used as an internal control. (b) Densitometric analysis of all samples was normalized against the level of total protein. The images are representative of the results of three independent experiments. All data are presented as the mean ± SD. ∗ P < 0.05.

Article Snippet: Antibodies for western blot analyses and the final working dilutions were as follows: rabbit polyclonal antibodies to α -SMA (dilution 1 : 1000; ABclonal, MA, USA), β -catenin (dilution 1 : 1000; ABclonal, MA, USA), vimentin (dilution 1 : 500; Santa Cruz, TX, USA), N-cadherin (dilution 1 : 1000; ABclonal, MA, USA), E-cadherin (dilution 1 : 1000; ABclonal, MA, USA), Wnt-3 (dilution 1 : 1000; ABclonal, MA, USA), p-GSK3 β (dilution 1 : 1000; ABclonal, MA, USA), GSK3 β (dilution 1 : 1000; ABclonal, MA, USA), β -actin (dilution 1 : 1000, Santa Cruz, TX, USA). p65 (dilution 1 : 1000; GeneTex, CA, USA), and fibrillin (dilution 1 : 1000, ABclonal, MA, USA).

Techniques: Expressing, Concentration Assay, SDS Page, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Role of Chrysophanol in Epithelial-Mesenchymal Transition in Oral Cancer Cell Lines via a Wnt-3-Dependent Pathway

doi: 10.1155/2020/8373715

Figure Lengend Snippet: The effect of the Wnt-3/pGSK3 β activator, Bml 284 on the expression of EMT markers, Wnt-3, pGSK3 β , and GSK3 β . (a) FaDu cells were treated with either the control (0 μ M, column 1) or the indicated concentration (0.7 μ M) of Bml 284 (column 2) in the presence of 30 μ M chrysophanol (Cho) for 24 h. Cells were then harvested and the proteins were separated by SDS-PAGE, followed by immunoblotting with the indicated antibodies. β -actin was used as an internal control. (b) Densitometric analysis of all samples normalized against the level of total protein. The images are representative of the results of three independent experiments. All data are presented as the mean ± SD. ∗ P < 0.05. ∗∗ P < 0.01.

Article Snippet: Antibodies for western blot analyses and the final working dilutions were as follows: rabbit polyclonal antibodies to α -SMA (dilution 1 : 1000; ABclonal, MA, USA), β -catenin (dilution 1 : 1000; ABclonal, MA, USA), vimentin (dilution 1 : 500; Santa Cruz, TX, USA), N-cadherin (dilution 1 : 1000; ABclonal, MA, USA), E-cadherin (dilution 1 : 1000; ABclonal, MA, USA), Wnt-3 (dilution 1 : 1000; ABclonal, MA, USA), p-GSK3 β (dilution 1 : 1000; ABclonal, MA, USA), GSK3 β (dilution 1 : 1000; ABclonal, MA, USA), β -actin (dilution 1 : 1000, Santa Cruz, TX, USA). p65 (dilution 1 : 1000; GeneTex, CA, USA), and fibrillin (dilution 1 : 1000, ABclonal, MA, USA).

Techniques: Expressing, Concentration Assay, SDS Page, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Role of Chrysophanol in Epithelial-Mesenchymal Transition in Oral Cancer Cell Lines via a Wnt-3-Dependent Pathway

doi: 10.1155/2020/8373715

Figure Lengend Snippet: A summary diagram outlines the working mechanisms of oridonin in FaDu cells. (a) Chrysophanol caused ROS accumulation. (b) Chrysophanol downregulated the expressions of Wnt-3 and nuclear translocations of NF- κ B/p65 or β -catenin. On the other hand, chrysophanol also attenuated EMT formation and cell migration.

Article Snippet: Antibodies for western blot analyses and the final working dilutions were as follows: rabbit polyclonal antibodies to α -SMA (dilution 1 : 1000; ABclonal, MA, USA), β -catenin (dilution 1 : 1000; ABclonal, MA, USA), vimentin (dilution 1 : 500; Santa Cruz, TX, USA), N-cadherin (dilution 1 : 1000; ABclonal, MA, USA), E-cadherin (dilution 1 : 1000; ABclonal, MA, USA), Wnt-3 (dilution 1 : 1000; ABclonal, MA, USA), p-GSK3 β (dilution 1 : 1000; ABclonal, MA, USA), GSK3 β (dilution 1 : 1000; ABclonal, MA, USA), β -actin (dilution 1 : 1000, Santa Cruz, TX, USA). p65 (dilution 1 : 1000; GeneTex, CA, USA), and fibrillin (dilution 1 : 1000, ABclonal, MA, USA).

Techniques: Migration

Journal: Annals of Translational Medicine

Article Title: miR-145-5p targets MMP2 to protect brain injury in hypertensive intracerebral hemorrhage via inactivation of the Wnt/β-catenin signaling pathway

doi: 10.21037/atm-22-1897

Figure Lengend Snippet: Effect of overexpression of miR-145-5p on the Wnt/β-catenin signaling pathway in thrombin-treated hBMECs. (A) Changes in mRNA expression of MMP2; (B) protein levels of MMP2, Wnt3, Wnt5a, β-catenin, TCF-4, LEF-1 and IL-8 in different groups detected by RT-PCR and western blot analysis, respectively. Results are presented as mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001. hBMECs, human brain microvascular endothelial cells; RT-PCR, reverse transcription-polymerase chain reaction; NC, negative control; SD, standard deviation; HICH, hypertensive intracerebral hemorrhage; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMP2, matrix metallopeptidase 2; TCF-4, transcription factor 4; LEF-1, lymphoid enhancer binding factor 1; IL-8, interleukin-8.

Article Snippet: The proteins were then transferred to a polyvinylidene fluoride membrane to be probed with Bax (Immunoway, 1:1,000), Bcl-2 (Immunoway, 1:1,000), Caspase 3 (Immunoway, 1:1,000), MMP2 (Immunoway, 1:1,000), Wnt3 (Immunoway, 1:1,000), Wnt5a (Immunoway, 1:1,000), β-catenin (Proteintech, 1:1,000), TCF-4 (Proteintech, 1:1,000), LEF-1(Proteintech, 1:1,000), IL-8 (Immunoway, 1:1,000) and GAPDH (Proteintech, 1:1,000) for 24 h at 4 °C.

Techniques: Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Negative Control, Standard Deviation, Binding Assay

Journal: Annals of Translational Medicine

Article Title: miR-145-5p targets MMP2 to protect brain injury in hypertensive intracerebral hemorrhage via inactivation of the Wnt/β-catenin signaling pathway

doi: 10.21037/atm-22-1897

Figure Lengend Snippet: Effect of miR-145-5p/MMP2 axis on the biological behavior of thrombin-treated hBMECs. (A) CCK8 assay of the proliferation of transfected hBMECs in each group; (B) flow cytometry to detect apoptosis of hBMECs in each group; (C) apoptosis-related proteins Bax, Bcl-2, and cleaved-caspase 3 determined by Western blot; (D) matrigel assay was used to assess the formation of capillary-like structures in hBMECs (400× magnification); (E) expression of ZO-1 and occludin was determined by immunofluorescence (400× magnification, scale bar: 50 µm); (F) vascular permeability was evaluated using FITC-labeled dextran. (G) Protein expression levels of MMP2, Wnt3, Wnt5a, β-catenin, TCF-4, LEF-1 and IL-8 in the 4 experimental groups. Results are presented as mean ± SD. **, P<0.01; ***, P<0.001. hBMECs, human brain microvascular endothelial cells; RT-PCR, reverse transcription-polymerase chain reaction; NC, negative control; SD, standard deviation; HICH, hypertensive intracerebral hemorrhage; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ZO-1, zonula occludens 1; FITC, fluorescein isothiocyanate; MMP2, matrix metallopeptidase 2; TCF-4, transcription factor 4; LEF-1, lymphoid enhancer binding factor 1; IL-8, interleukin-8.

Article Snippet: The proteins were then transferred to a polyvinylidene fluoride membrane to be probed with Bax (Immunoway, 1:1,000), Bcl-2 (Immunoway, 1:1,000), Caspase 3 (Immunoway, 1:1,000), MMP2 (Immunoway, 1:1,000), Wnt3 (Immunoway, 1:1,000), Wnt5a (Immunoway, 1:1,000), β-catenin (Proteintech, 1:1,000), TCF-4 (Proteintech, 1:1,000), LEF-1(Proteintech, 1:1,000), IL-8 (Immunoway, 1:1,000) and GAPDH (Proteintech, 1:1,000) for 24 h at 4 °C.

Techniques: CCK-8 Assay, Transfection, Flow Cytometry, Western Blot, Matrigel Assay, Expressing, Immunofluorescence, Permeability, Labeling, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Polymerase Chain Reaction, Negative Control, Standard Deviation, Binding Assay

Journal: Annals of Translational Medicine

Article Title: miR-145-5p targets MMP2 to protect brain injury in hypertensive intracerebral hemorrhage via inactivation of the Wnt/β-catenin signaling pathway

doi: 10.21037/atm-22-1897

Figure Lengend Snippet: Effect of miR-145-5p in HICH rat model. (A) The protein expression levels of ZO-1 and occludin were determined by immunofluorescence (scale bar: 50 µm); (B) the protein levels of MMP2, Wnt3, Wnt5a, β-catenin, TCF-4, LEF-1 and IL-8 in different groups detected by western blot analysis. *, P<0.05; ***, P<0.001. HICH, hypertensive intracerebral hemorrhage; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride.

Article Snippet: The proteins were then transferred to a polyvinylidene fluoride membrane to be probed with Bax (Immunoway, 1:1,000), Bcl-2 (Immunoway, 1:1,000), Caspase 3 (Immunoway, 1:1,000), MMP2 (Immunoway, 1:1,000), Wnt3 (Immunoway, 1:1,000), Wnt5a (Immunoway, 1:1,000), β-catenin (Proteintech, 1:1,000), TCF-4 (Proteintech, 1:1,000), LEF-1(Proteintech, 1:1,000), IL-8 (Immunoway, 1:1,000) and GAPDH (Proteintech, 1:1,000) for 24 h at 4 °C.

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: Aging (Albany NY)

Article Title: Identification of candidate lncRNAs and circRNAs regulating WNT3/β-catenin signaling in essential hypertension

doi: 10.18632/aging.103137

Figure Lengend Snippet: Co-expression subnetwork of DE lncRNAs associated with WNT3 and CAMK2N2. ( A ) Co-expression network analysis indicating positive correlation between 7 DE lncRNAs and both WNT3 and CAMK2N2. ( B ) A positive correlation with both WNT3 and CAMK2N2 was detected for 14 circRNAs. We used the circBase database for gene annotation; only one gene was not annotated in circBase. ( C , D ) A miRNA-based sponge regulatory subnetwork depicting LOC646616/miRNA/WNT3 and LOC646616/miRNA/CAMK2N2 interactive modules.

Article Snippet: Primary antibodies against WNT3 (cat. GTX89319; Gene Tex, CA, USA), β-catenin (cat. GTX101435-S; Gene Tex), calcium/calmodulin-dependent protein kinase II inhibitor 2 (CAMK2N2, cat. TA322113S; Origene, MD, USA), and GAPDH (cat. TA-08; ZSGB-BIO, Beijing, China) were applied at 4 °C overnight.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Identification of candidate lncRNAs and circRNAs regulating WNT3/β-catenin signaling in essential hypertension

doi: 10.18632/aging.103137

Figure Lengend Snippet: WNT3 interacts directly with miR-637. ( A ) qRT-PCR analysis of relative WNT3 and β-catenin expression in PBMNCs of hypertensive subjects and normotensive controls. ( B ) Western blot analysis of WNT3 and β-catenin levels in plasma of hypertensive subjects and normotensive controls. ( C ) Bioinformatics evidence of the interaction between miR-637 and the 3′-UTR of WNT3. Bottom: schematic diagram of the mutations in the WNT3 sequence used to create the mutant luciferase reporter construct. ( D ) Luciferase activity assay in HEK293T cells co-transfected with miR-637 mimics or mimic NC and luciferase report plasmids containing wild type (wt) or mutant (mut) WNT3 3′ UTR and. ( E ) RIP assay results showing co-precipitation of miR-637 and WNT3 mRNA complexes by an Ago2 antibody in HASMCs. Validation data obtained by qRT-PCR are also shown. ( F ) Western blot analysis of WNT3 levels in HASMCs transfected with miR-637 mimics or mimic NC. Expression data are normalized to GAPDH. ( G ) Spearman's correlation analysis of the relationship between LOC646616, WNT3, and miR-637 levels, and between WNT3 and β-catenin levels in our hypertensive cohort. Data are presented as the mean ± SD. n = 3 biologically independent samples. * P <0.05, ** P <0.01.

Article Snippet: Primary antibodies against WNT3 (cat. GTX89319; Gene Tex, CA, USA), β-catenin (cat. GTX101435-S; Gene Tex), calcium/calmodulin-dependent protein kinase II inhibitor 2 (CAMK2N2, cat. TA322113S; Origene, MD, USA), and GAPDH (cat. TA-08; ZSGB-BIO, Beijing, China) were applied at 4 °C overnight.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Clinical Proteomics, Sequencing, Mutagenesis, Luciferase, Construct, Activity Assay, Transfection, Biomarker Discovery

Journal: Aging (Albany NY)

Article Title: Identification of candidate lncRNAs and circRNAs regulating WNT3/β-catenin signaling in essential hypertension

doi: 10.18632/aging.103137

Figure Lengend Snippet: LOC646616 increases WNT3 expression by sponging miR-637. ( A ) qRT-PCR analysis of LOC646616 expression in HASMCs transfected with control siRNA (si-NC) or with 3 siRNA variants targeting LOC646616. ( B , C ) Results of Transwell invasion and viability (CCK-8) assays conducted in HASMCs transfected with si-LOC646616-2. ( D ) Left panel: relative miR-637 expression in HASMCs transfected with si-NC or si-LOC646616-2. Right panel: relative WNT3 mRNA expression in HASMCs transfected with NC or anti-miR-637. ( E , F ) WNT3 mRNA (left panel) and protein (middle and right panel) levels in HASMCs following knockdown of LOC646616 (si-LOC-2) with/without concurrent inhibition of miR-637. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Primary antibodies against WNT3 (cat. GTX89319; Gene Tex, CA, USA), β-catenin (cat. GTX101435-S; Gene Tex), calcium/calmodulin-dependent protein kinase II inhibitor 2 (CAMK2N2, cat. TA322113S; Origene, MD, USA), and GAPDH (cat. TA-08; ZSGB-BIO, Beijing, China) were applied at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, CCK-8 Assay, Knockdown, Inhibition

Journal: Aging (Albany NY)

Article Title: Identification of candidate lncRNAs and circRNAs regulating WNT3/β-catenin signaling in essential hypertension

doi: 10.18632/aging.103137

Figure Lengend Snippet: Schematic representation of the LOC646616/miR-637/WNT3 ceRNA network in EH. LOC646616 indirectly promotes WNT3 mRNA translation by sponging miR-637, leading to WNT3/β-catenin pathway activation.

Article Snippet: Primary antibodies against WNT3 (cat. GTX89319; Gene Tex, CA, USA), β-catenin (cat. GTX101435-S; Gene Tex), calcium/calmodulin-dependent protein kinase II inhibitor 2 (CAMK2N2, cat. TA322113S; Origene, MD, USA), and GAPDH (cat. TA-08; ZSGB-BIO, Beijing, China) were applied at 4 °C overnight.

Techniques: Activation Assay