fto polyclonal antibody  (Proteintech)

Journal: Non-coding RNA Research

Article Title: circ_0103896/miR-432–5p/FTO feedback loop suppresses the formation and progression of intracranial aneurysm

doi: 10.1016/j.ncrna.2025.11.001

Figure Lengend Snippet: FTO is regulated by the circ_0103896/miR-432–5p axis in hBVSMCs. ( A ) Schematic highlighting the predicted binding sites of miR-432–5p within the 3′UTR of FTO. ( B ) Luciferase essay in hBVSMCs comparing WT and mutant FTO 3′UTRs following miR-432–5p mimic transfection. ∗∗∗ P < 0.001. ( C-E ) FTO expression in hBVSMCs after transfection with circ_0103896 overexpression vectors or miR-432–5p mimics, assessed by RT-qPCR ( C ) and Western blot ( D, E ). ∗∗∗ P < 0.001. ( F-I ) Protein expression of α-SMA ( F, G ), SM22α ( F, G ), and MMP2 and MMP9 ( H, I ), in hBVSMCs transfected with circ_0103896 or miR-432–5p mimics. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. ( J-N ) Assessment of hBVSMC proliferation ( J ), migration ( K, L ), and apoptosis ( M, N ) after transfection with FTO plasmids or miR-432–5p mimics, using MTT assay, Transwell migration, and cell apoptosis analysis. ∗∗ P < 0.01 and ∗∗∗ P < 0.001. Data are represented as mean ± SEM from ≥3 independent biological replicates. qPCR and luciferase assays were performed in technical triplicate. Statistical comparisons were conducted using an unpaired Student's t -test for two-group comparisons or one-way ANOVA with Tukey's post hoc test for multiple groups. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: All antibodies were purchased from Cell Signaling Technology (USA): FTO (45980S, 1:1000), α-SMA (14968S, 1:1000), SM22α (52011S, 1:1000), MMP2 (14351S, 1:1000), MMP9 (24317S, 1:1000), and GAPDH (2118L, 1:1000).

Techniques: Binding Assay, Luciferase, Mutagenesis, Transfection, Expressing, Over Expression, Quantitative RT-PCR, Western Blot, Migration, MTT Assay

Journal: Non-coding RNA Research

Article Title: circ_0103896/miR-432–5p/FTO feedback loop suppresses the formation and progression of intracranial aneurysm

doi: 10.1016/j.ncrna.2025.11.001

Figure Lengend Snippet: FTO mediates m 6 A modification of circ_0103896, establishing a positive feedback loop that reinforces circ_0103896 expression. ( A, B ) Global RNA m 6 A levels in hBVSMCs after FTO overexpression or silencing, measured by MeRIP-qPCR ( A ) and m 6 A dot-blot assay ( B ). Dot-blot signals were normalized to methylene blue staining. ∗∗ P < 0.01. ( C ) MeRIP analysis of circ_0103896 m 6 A modification in hBVSMCs following FTO overexpression or silencing. ∗∗ P < 0.01. ( D ) RT-qPCR measurement of circ_0103896 expression in hBVSMCs after FTO overexpression or silencing. ∗ P < 0.05 and ∗∗∗ P < 0.001. ( E ) RIP analysis showing binding between FTO and circ_0103896 in hBVSMCs. ∗∗∗ P < 0.001. ( F, G ) Representative western blots ( F ) and quantification ( G ) blots of α-SMA and SM22α protein levels after indicated transfections. ∗∗∗ P < 0.001. ( H, I ) Representative western blots ( H ) and quantification ( I ) of MMP2 and MMP9 protein levels in hBVSMCs after indicated transfections. ∗∗∗ P < 0.001. ( J ) MTT assay assessing hBVSMC proliferation following circ_0103896 silencing, FTO overexpression, or combined treatments. ∗∗ P < 0.01 and ∗∗∗ P < 0.001. ( K, L ) Representative images ( K ) and quantification ( L ) of hBVSMC migration after circ_0103896 silencing, FTO overexpression, or combined treatments. ∗∗∗ P < 0.001. ( M, N ) Representative histograms ( M ) and quantification ( N ) of apoptosis in hBVSMCs after circ_0103896 silencing, FTO overexpression, or combined treatments. ∗∗ P < 0.01 and ∗∗∗ P < 0.001. Data are presented as mean ± SEM from ≥3 independent biological replicates. MeRIP and dot-blot assays were performed in technical triplicate for each sample. Comparisons were analyzed using one-way ANOVA with Tukey's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: All antibodies were purchased from Cell Signaling Technology (USA): FTO (45980S, 1:1000), α-SMA (14968S, 1:1000), SM22α (52011S, 1:1000), MMP2 (14351S, 1:1000), MMP9 (24317S, 1:1000), and GAPDH (2118L, 1:1000).

Techniques: Modification, Expressing, Over Expression, Dot Blot, Staining, Quantitative RT-PCR, Binding Assay, Western Blot, Transfection, MTT Assay, Migration

Journal: Non-coding RNA Research

Article Title: circ_0103896/miR-432–5p/FTO feedback loop suppresses the formation and progression of intracranial aneurysm

doi: 10.1016/j.ncrna.2025.11.001

Figure Lengend Snippet: circ_0103896 suppresses IA formation and progression in vivo via the circ_0103896/miR-432–5p/FTO feedback loop. ( A ) Representative H&E-stained images illustrating vessel wall integrity in IA mice under different treatments. ( B ) Representative bar graph depicting the aneurysmal scores of IA mice under differential treatments. ∗∗ P < 0.01 and ∗∗∗∗ P < 0.0001. ( C ) Pie charts depicting the distribution of IA grades across treatment groups. ( D-G ) Representative bar graphs showing mRNA expression of inflammatory mediators, TNF-α ( D ), IL1B ( E ), MMP9 ( F ), and MMP2 ( G ), in cerebral arteries of differentially treated IA mice. ∗∗∗ P < 0.001. Each mouse represents one biological replicate ( n = 6 per group unless otherwise indicated). Data are presented as mean ± SEM. Aneurysmal scores and cytokine expression were compared using one-way ANOVA with Tukey's post hoc test; The IA grade distributions were analyzed by chi-square test. ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001. Mice were randomly assigned to groups using a computer-generated sequence stratified by baseline body weight. Histological scoring, grade assignment, and cytokine quantification were performed on coded samples by investigators blinded to group allocation. Group sizes ( n = 6 per group) were determined a priori based on power calculation for the aneurysmal score (α = 0.05, power = 0.8, expected mean difference = 1.3, SD = 0.8).

Article Snippet: All antibodies were purchased from Cell Signaling Technology (USA): FTO (45980S, 1:1000), α-SMA (14968S, 1:1000), SM22α (52011S, 1:1000), MMP2 (14351S, 1:1000), MMP9 (24317S, 1:1000), and GAPDH (2118L, 1:1000).

Techniques: In Vivo, Staining, Expressing, Generated, Sequencing

Journal: Europace

Article Title: Fto-mediated m 6 A demethylation of Lox drives atrial fibrosis and promotes atrial fibrillation in a murine model of hyperthyroidism

doi: 10.1093/europace/euag047

Figure Lengend Snippet: Cardiomyocyte-specific deletion of Fto alleviated T4-induced AF. ( A ) Representative traces of AF induced by electrical stimulation in Myh6-cre − ; Fto fl/fl mice and Myh6-cre + ; Fto fl/fl mice in saline and T4 groups. ( B ) Quantification of AF inducibility ( n = 10). ( C ) AF duration was quantified only in animals with inducible episodes. The sample size for each group is n = 10, of which 2 (Myh6-cre − ; Fto fl/fl ) and 7 (Myh6-cre + ; Fto fl/fl ) animals did not have inducible AF and are therefore not included in (C) . ( D ) Quantification of HW/TL ( n = 10). ( E ) Representative maps of atrial activation time and dispersion of conduction in Myh6-cre − ; Fto fl/fl mice and Myh6-cre + ; Fto fl/fl mice in saline and T4 groups ( n = 6). ( F ) Quantification of active time ( n = 6). ( G ) Quantification of dispersion of conduction ( n = 6). ( H ) Quantification of LACV ( n = 6). ( I ) Quantification of ERP ( n = 6). ( J ) Representative images of LAD and Masson staining in atria. ( K ) Quantification of fibrosis area ( n = 6). ( L ) Quantification of LAD ( n = 6). AF, atrial fibrillation; β-Actin, beta-actin; ERP, effective refractory period; Fto, fat mass and obesity-associated protein; HR, heart rate; HW/TL, heart weight-to-tibial length ratio; LACV, left atrial conduction velocity; LAD, left atrial diameter. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Statistical analysis was performed with ordinary one-way ANOVA ( D , F , G , H , I , K , and L ), Fisher's exact test ( B ) and unpaired Student's t -test ( C ).

Article Snippet: Cardiomyocyte-specific Fto knockout mice ( Myh6-Cre + ; Fto fl/fl ) were established on a C57BL/6J background (Shanghai Model Organisms, NM-CKO-190005) and analysed at 2 months of age.

Techniques: Saline, Activation Assay, Dispersion, Staining

Journal: Europace

Article Title: Fto-mediated m 6 A demethylation of Lox drives atrial fibrosis and promotes atrial fibrillation in a murine model of hyperthyroidism

doi: 10.1093/europace/euag047

Figure Lengend Snippet: Cardiomyocyte-specific Fto knockout attenuated Lox expression in T4-treated mice. ( A and B ) GO functional enrichment analysis of differentially expressed genes. ( C ) Venn diagram showing overlap of differentially expressed genes between two groups. ( D ) Representative immunoblots of Fto and Lox protein in Myh6-cre − ; Fto fl/fl mice and Myh6-cre + ; Fto fl/fl mice in saline and T4 groups ( n = 6). ( E ) Quantification of Lox mRNA levels ( n = 6). ( F ) Quantification of Fto protein levels ( n = 6). ( G ) Quantification of Lox protein levels ( n = 6). β-Actin, beta-actin; Fto, fat mass and obesity-associated protein; GO, gene ontology; Lox, lysyl oxidase. **** P < 0.0001, comparison made using ordinary one-way ANOVA.

Article Snippet: Cardiomyocyte-specific Fto knockout mice ( Myh6-Cre + ; Fto fl/fl ) were established on a C57BL/6J background (Shanghai Model Organisms, NM-CKO-190005) and analysed at 2 months of age.

Techniques: Knock-Out, Expressing, Functional Assay, Western Blot, Saline, Comparison

Journal: Europace

Article Title: Fto-mediated m 6 A demethylation of Lox drives atrial fibrosis and promotes atrial fibrillation in a murine model of hyperthyroidism

doi: 10.1093/europace/euag047

Figure Lengend Snippet: Cardiomyocyte-specific Fto overexpression promoted AF susceptibility via m 6 A-dependent mechanisms. ( A ) Representative immunoblots of Fto and Lox proteins in mice injected with AAV9-cTnT- Fto wt -oe, AAV9-cTnT- Fto mut -oe, and AAV9-cTnT-nc ( n = 6). ( B and C ) Quantification of Fto and Lox protein levels ( n = 6). ( D ) Representative intracardiac bipolar electrograms showing induction of AF from mice injected with AAV9-cTnT- Fto wt -oe, AAV9-cTnT- Fto mut -oe, and AAV9-cTnT-nc. ( E ) Quantification of AF inducibility ( n = 10). ( F ) AF was inducible only in the AAV9-cTnT- Fto wt -oe group (7 out of 10 animals). Therefore, data are presented only for this group. AF was not inducible in any animal from the AAV9-cTnT-nc ( n = 10) or AAV9-cTnT- Fto mut -oe ( n = 10) groups; hence, no AF duration data are available for these groups, and they are not represented in this graph. ( G ) Representative maps of atrial activation time and dispersion of conduction. ( H ) Quantification of active time ( n = 6). ( I ) Quantification of dispersion of conduction ( n = 6). ( J ) Quantification of LACV ( n = 6). ( K ) Quantification of ERP ( n = 6). ( L ) Representative images of LAD and Masson staining. ( M ) Quantification of LAD ( n = 6). ( N ) Quantification of percentage of fibrotic area ( n = 6). AF, atrial fibrillation; β-Actin, beta-actin; ERP, effective refractory period; Fto, fat mass and obesity-associated protein; ; LACV, left atrial conduction velocity; LAD, left atrial diameter; Lox, lysyl oxidase. ** P < 0.01; *** P < 0.001; **** P < 0.0001. Statistical analysis was performed with ordinary one-way ANOVA ( B , C , H , I , J , K , M , and N ), Fisher's exact test ( E ).

Article Snippet: Cardiomyocyte-specific Fto knockout mice ( Myh6-Cre + ; Fto fl/fl ) were established on a C57BL/6J background (Shanghai Model Organisms, NM-CKO-190005) and analysed at 2 months of age.

Techniques: Over Expression, Western Blot, Injection, Activation Assay, Dispersion, Staining