Journal: Scientific reports

Article Title: WNTA5-mediated miR-374a-5p regulates vascular smooth muscle cell phenotype transformation and M1 macrophage polarization impacting intracranial aneurysm progression.

doi: 10.1038/s41598-024-51243-z

Figure Lengend Snippet: Figure 3. miR-374a-5p targeted WNT signaling (A) qRT-PCR detection of WNT5A, WNT3, and WNT2B expression. (B) WB detection of WNT5A, WNT3, and WNT2B expression. *P < 0.05 vs. Sham. (C) Pearson correlation analysis of the expression of miR-374a-5p and WNT5A, WNT3, and WNT2B. (D) WB detection of WNT5A expression. *P < 0.05 vs. Sham, &P < 0.05 vs. IA + inhibitor NC. (E) Bioinformatics prediction of miR- 374a-5p targeting of WNT signaling (WNT2B, WNT3, and WNT5A). (F) To validate the targeting relationship between miR-374a-5p and WNTA5, a dual luciferase reporter assay was conducted. *P < 0.05 vs. miR-374a-5p NC. n = 3.

Article Snippet: Indicator Dilution Origin Manufacturer Calponin 1:5000 Rabbit 24855-1-AP, Proteintech TIMP3 1:1000 Rabbit 10858-1-AP, Proteintech TIMP4 1:1000 Rabbit 12326-1-AP, Proteintech SM22α 1:5000 Rabbit ab155272, Abcam α-SMA 1 μg/mL Rabbit ab5694, Abcam MMP1 1:1000 Rabbit 26585-1-AP, Proteintech MMP3 1:1000 Rabbit ab53015, Abcam MMP9 1:1000 Rabbit 27306-1-AP, Proteintech WNT2B 1:3000 Rabbit ab178418, Abcam WNT3 1 μg/mL Rabbit ab32249, Abcam WNT5A 1:3000 Rabbit ab235966, Abcam GAPDH 1:5000 Rabbit 10494-1-AP, Proteintech HRP goat anti-mouse IgG 1:5000 Mouse SA00001-1, Proteintech HRP goat anti-rabbit IgG 1:6000 Rabbit SA00001-2, Proteintech 5 Vol.

Techniques: Quantitative RT-PCR, Expressing, Luciferase, Reporter Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Berberine Protects C17.2 Neural Stem Cells From Oxidative Damage Followed by Inducing Neuronal Differentiation

doi: 10.3389/fncel.2019.00395

Figure Lengend Snippet: Real time PCR primers.

Article Snippet: Antibodies for detecting the following proteins were used in this research: NRF-1 (Santa Cruz, sc-515360), NRF-2 (Santa Cruz, sc-365949), NQO-1 (Santa Cruz, sc-32793), HO-1 (Santa Cruz, sc-136960), Bax (Santa Cruz, sc-20067), Bcl2 (Santa Cruz, sc-23960), Caspase 3 (Cell Signaling, 9662S), Cleaved Caspase 3 (Cell Signaling, 9661S), ERK1/2 (Santa Cruz, sc-514302), pERK (Santa Cruz, sc-377400), Nestin (Santa Cruz, sc-23927), MAP2 (Santa Cruz, sc-74421), Olig 2 (Abcam, ab109186), GFAP (Santa Cruz, sc-33673), WNT3α (Santa Cruz, sc-74537), β-Catenin (Santa Cruz, sc-53483), β-Actin (Santa Cruz, sc-47778), TUBB3 (Santa Cruz, sc-80016), ASCL1 (Santa Cruz, sc-374104), NeuroG1 (Santa Cruz, sc-100332), NeuroD2 (Santa Cruz, sc-365896) and DCX (Santa Cruz, sc-271390).

Techniques: Real-time Polymerase Chain Reaction

Journal: Frontiers in Cellular Neuroscience

Article Title: Berberine Protects C17.2 Neural Stem Cells From Oxidative Damage Followed by Inducing Neuronal Differentiation

doi: 10.3389/fncel.2019.00395

Figure Lengend Snippet: Differentiation pathway. (A,B) mRNA levels of WNT3α (A) and β-Catenin (B) in Group 1, 2, 3, and 4. (C) Western blot analysis of WNT3a and b-Catenin in Group 1, 2, 3, and 4. (D,E) Quantification of protein blots. Group 1 was set as the control. The data represent the mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Antibodies for detecting the following proteins were used in this research: NRF-1 (Santa Cruz, sc-515360), NRF-2 (Santa Cruz, sc-365949), NQO-1 (Santa Cruz, sc-32793), HO-1 (Santa Cruz, sc-136960), Bax (Santa Cruz, sc-20067), Bcl2 (Santa Cruz, sc-23960), Caspase 3 (Cell Signaling, 9662S), Cleaved Caspase 3 (Cell Signaling, 9661S), ERK1/2 (Santa Cruz, sc-514302), pERK (Santa Cruz, sc-377400), Nestin (Santa Cruz, sc-23927), MAP2 (Santa Cruz, sc-74421), Olig 2 (Abcam, ab109186), GFAP (Santa Cruz, sc-33673), WNT3α (Santa Cruz, sc-74537), β-Catenin (Santa Cruz, sc-53483), β-Actin (Santa Cruz, sc-47778), TUBB3 (Santa Cruz, sc-80016), ASCL1 (Santa Cruz, sc-374104), NeuroG1 (Santa Cruz, sc-100332), NeuroD2 (Santa Cruz, sc-365896) and DCX (Santa Cruz, sc-271390).

Techniques: Western Blot, Control

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: 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