fto  (Cell Signaling Technology Inc)

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: Cell Research

Article Title: The AHCY–adenosine complex rewires mRNA methylation to enhance fatty acid biosynthesis and tumorigenesis

doi: 10.1038/s41422-025-01213-5

Figure Lengend Snippet: a Two representative 32 P autoradiograms from CLIP of FTO-HA pulled down from AHCY KO HCT116 cells re-expressing AHCY WT or the indicated mutants using a monoclonal anti-HA antibody ( n = 6). b Purified recombinant His-FTO and GST-AHCY WT or the indicated mutants (5 μg) were incubated overnight with the biotin-m 6 A motif fragment (200 nM) in the presence or absence of 10 μM ADO. Pulldown assays were performed with streptavidin beads. c Demethylation of m 6 A in the post-FTO reaction mixtures in vitro. With H 2 O as a mock control, purified recombinant AHCY or the indicated mutant was added to the standard reaction mixture for FTO demethylation in the presence or absence of 10 μM ADO, and the reaction products were then analyzed by LC-MS/MS. d Alignment of the FTO–ssDNA complex (PDB: 5ZMD) and the FTO–AHCY dimer complex in Fig. . FTO proteins, ssDNA, and AHCY proteins are shown as cartoons and are colored purple, yellow, and green, respectively. e Western blot analysis of co-immunoprecipitation of FTO-Flag or the indicated mutants with endogenous AHCY in the indicated HCT116 and A549 cells. f Significantly decreased (red dot) or increased (black dot) FTO binding peaks were identified in AHCY-depleted A549 cells upon AHCY re-expression (false discovery rate (FDR) < 0.0001 and FC > 2). g A density plot shows the distribution of motifs enriched in the Vector group from FTO CLIP-seq, together with their respective distances from the m 6 A motif DRACH. Each color corresponds to a motif. h Motifs harboring the DRACH sequence identified by HOMER de novo analysis from FTO CLIP-seq peaks enriched in the Vector group. i Purified recombinant FTO (5 μg) and AHCY (5 μg) proteins were incubated overnight with different sequences of biotin-GG(m 6 A)C motif fragments (200 nM; GGGGAC, TGGGAC, CAGGAC, or GAGGAC). Pull-down assays were performed with streptavidin beads. j Purified recombinant FTO (5 μg) and the indicated mutant proteins (5 μg) were incubated overnight with different sequences of biotin-GG(m 6 A)C motif fragments (200 nM). k , l AHCY-depleted A549 or SW480 cells re-expressing WT or mutant AHCY were treated with or without 25 μM ADO for 12 h. Relative precipitated RNA levels were normalized to those in non-ADO-treated cells expressing WT AHCY. m AHCY-depleted SW480 and A549 cells re-expressing WT or mutant AHCY with or without FTO shRNA transduction. Relative precipitated RNA levels were normalized to those in cells expressing WT AHCY. RNA immunoprecipitation assays were performed in these cells using anti-FTO ( k ) or anti-m 6 A ( l , m ) antibodies, and qPCR analysis of precipitated ACACA mRNA was then performed. n The mRNA levels of ACACA in AHCY-depleted SW480 and A549 cells re-expressing WT or mutant AHCY with or without FTO shRNA transduction were examined by qPCR. o Schematic diagram depicting the incorporation of carbons from uniformly-labeled 13 C-glucose (U-13C-glucose, indicated by the black filled circles) into the TCA cycle, as well as palmitate, stearate, palmitoleate, and oleate. Representative palmitate isotopologs are depicted. p Steady-state palmitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), and oleic acid (18:1) labeling from U- 13 C-glucose in the indicated A549 tumor cells cultured in lipid-free CM for 24 h. FA taken up exogenously were also incorporated into cellular FFAs but were not labeled. q Quantification of malonyl-CoA in AHCY-depleted HCT116 and A549 cells re-expressing WT AHCY or the indicated mutants. r GC-MS/MS quantification of the normalized abundances of FFAs in AHCY-depleted A549 and SW480 cells re-expressing WT or mutant AHCY cultured in lipid-free CM or regular CM ( n = 6). s The ratios of MUFAs (C16:1 and C18:1) to SFAs (C16:0 and 18:0), representing SCD1 activity in AHCY-depleted A549 and SW480 cells re-expressing WT or mutant AHCY cultured in lipid-free CM or regular CM, as measured by GC-MS/MS ( n = 6). Data are prese n ted as mean ± S.D. ( n = 3, unless otherwise specified). One-way ANOVA with LSD- t ( a , c , k – n , p – s ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, N.S., not significant.

Article Snippet: The antibodies used in this study were anti-AHCY (Proteintech, 10757-2-AP, 1:1000), anti-MAT2A (CST, 84478, 1:1000), anti-Histone H3 (CST, 4499, 1:2000), anti-H3K4me3 (CST, 9751, 1:2000), anti-H3K9me3 (CST, 13969, 1:2000), anti-H3K27me3 (CST, 9733, 1:2000), anti-H3K36me3 (CST, 4909, 1:2000), anti-H3K79me3 (Abways, AB3548, 1:1000), anti-FTO (CST, 45980, 1:1000), anti-actin (CST, 93473, 1:1000), anti-GAPDH (CST, 92310, 1:1000), anti-PARP (CST, 9542, 1:1000), anti-flag (Sigma, F3165, 1:1000), anti-HA (CST, 2367, 1:1000), anti-Myc (CST, 5605, 1:1000), anti-SAHH (Abcam, ab134966, 1:1000), anti-ACC1 (Proteintech, 21923-1-AP, 1:1000), anti-GST (Proteintech, 66001-2-Ig, 1:2000), FASN (Acmec, AC50671, 1:1000), anti-SCD1 (Proteintech, 28678-1-AP, 1:1000), anti-SREBP1 (Santa Cruz, sc-13551, 1:1000), SREBP2 (Santa Cruz, sc-13552, 1:1000), anti-p-AMPK (CST, 9957, 1:1000), anti-AMPK (CST, 9957, 1:1000), anti-p-PI3K (GeneTex, GTX100462S, 1:1000), anti-Akt (CST, 927, 1:10002), anti-EGFR (Proteintech, 66455-1-Ig, 1:1000), anti-Ki67 (Proteintech, 27309-1-AP, 1:1000), anti-ALKBH5 (Proteintech, 16837-1-AP, 1:1000), anti-METTL3 (Proteintech, 15073-1-AP, 1:1000), and anti-METTL14 (Proteintech, 26158-1-AP, 1:1000).

Techniques: Expressing, Purification, Recombinant, Incubation, In Vitro, Control, Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Western Blot, Immunoprecipitation, Binding Assay, Plasmid Preparation, Sequencing, shRNA, Transduction, RNA Immunoprecipitation, Labeling, Cell Culture, Gas Chromatography-Mass Spectrometry, Activity Assay

Journal: BMC Immunology

Article Title: FTO-mediated m6A demethylation of BECN1 mRNA promotes hepatic steatosis by impairing autophagy

doi: 10.1186/s12865-025-00784-7

Figure Lengend Snippet: FTO interacts with BECN1 mRNA and modulates its m6A methylation. (A) Relative mRNA expression of autophagy-related genes (ATG5, ATG7, BECN1, BNIP3, and LAMP1) in FTO-knockdown HepG2 cells, as determined by qPCR. (B) Relative m6A levels of autophagy-related genes were measured by Me-RIP. (C) Interaction between FTO and BECN1 mRNA in HepG2 cells, as assessed by RIP assay. (D) Prediction score distribution along the BECN1 mRNA sequence for potential m6A modification sites using the SRAMP database. (E-G) Relative luciferase activity of WT and MUT BECN1 constructs in FTO-knockdown HepG2 cells, as determined by luciferase reporter assays. (H) Stability of BECN1 mRNA in FTO-knockdown HepG2 cells over time, as determined by qPCR. Data are presented as mean ± SD; ** p < 0.01 vs. shNC or IgG group. ( n = 3)

Article Snippet: The lysate was incubated with magnetic beads conjugated with 5 μg of anti-FTO antibody (45980, CST) or normal rabbit IgG (ab133470, Abcam) as a negative control, for 6 h at 4 °C with rotation.

Techniques: Methylation, Expressing, Knockdown, Sequencing, Modification, Luciferase, Activity Assay, Construct