Journal: Journal of Cancer

Article Title: N6-methyladenosine regulates ATM expression and downstream signaling.

doi: 10.7150/jca.64061

Figure Lengend Snippet: Figure 2. METTL3 and FTO modulate ATM expression. (A) BJAB cells were treated with 10 μM 3-DAA (control group were treated with equal amount of PBS) for 2 h, cellular RNA was extracted at 24 h and mRNA expression of ATM was assayed by RT-qPCR (n=3). Western blot assay was used to detect ATM protein expression. (B) Western blot assay was used to detect ATM and METTL3 protein expression levels in sh-NC or sh-METTL3 cells. (C) RT-qPCR was used to detect ATM and METTL3 mRNA expression levels in sh-NC or sh-METTL3 BJAB cells. (D) BJAB cells (sh-NC and sh-METTL3) were treated with Act-D (5 μg/ml) for indicated times, then, RT-qPCR assay was performed to detect ATM mRNA expressions. Mean ± SD of three independent experiments. Two-tailed unpaired t-test. (E) SGC7901 cells were transfected with indicated siRNAs for 24 h. ATM mRNA expression was detected by RT-qPCR. (F) SGC7901 cells (si-NC and si-FTO) were treated with Act-D (5 μg/ml) for indicated times, then, RT-qPCR assay was performed to detect ATM mRNA expressions. (G) SGC7901 cells were treated with indicated siRNAs for 48 h, Western blot was used to detect the indicated proteins. *p < 0.05, **p < 0.01, ***p < 0.001. ns, no significant difference.

Article Snippet: Protein extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel, transferred to polyvinylidene fluoride membranes, and probed with antibodies against ATM (2873S, Cell Signaling Technology, CST); p-ATM -S1981 (AP0008, ABclonal); METTL3 (15073-1-AP, Proteintech); YTHDF1 (17479-1-AP, Proteintech); YTHDF2 (24744-1-AP, Proteintech); YTHDF3 (25537-1-AP, Proteintech); FTO (TA809392, OriGene); m6A (202003, Synaptic Systems); BRCA1 (20649-1-AP, Proteintech); H2A.X (A11361, ABclonal); γH2A.X (AP0687, ABclonal ); GAPDH(60004-1-Ig, Proteintech); eIF3A (3411T, CST); Flag (F1804, Sigma-Aldrich); anti‐rabbit IgG HRP‐ linked antibody (7074; CST); Peroxidase-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (SA00001-1, Proteintech); α-Tubulin (66031-1-Ig, Proteintech); CHK1(bs-1681R, Bioss); CHK2 (252092, Zenbio); p-CHK2-S19(AP0862, ABclonal); Anti-HA tag monoclonal antibody (TA100012, OriGene); Mouse-IgG (BA1046, Boster); Rabbit IgG (B900610, Proteintech); p21(10355-1-AP, Proteintech); 53BP1 (BA2878, Boster); CDK1 (D160158, BBI life sciences); CDK2 (D199431, BBI life sciences); Cyclin B2 (21644-1-AP, Proteintech).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Two Tailed Test, Transfection

Journal: Journal of Cancer

Article Title: N6-methyladenosine regulates ATM expression and downstream signaling.

doi: 10.7150/jca.64061

Figure Lengend Snippet: Figure 6. Hypothetical model of the mechanism how m6A modification regulates ATM expression and downstream signaling. METTL3 increases the m6A modification of ATM and inhibits ATM expression and mRNA stability, whereas FTO functions oppositely. YTHDF1/2 bind to the m6A sites and recruit eIF3A, leading ATM degradation.

Article Snippet: Protein extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel, transferred to polyvinylidene fluoride membranes, and probed with antibodies against ATM (2873S, Cell Signaling Technology, CST); p-ATM -S1981 (AP0008, ABclonal); METTL3 (15073-1-AP, Proteintech); YTHDF1 (17479-1-AP, Proteintech); YTHDF2 (24744-1-AP, Proteintech); YTHDF3 (25537-1-AP, Proteintech); FTO (TA809392, OriGene); m6A (202003, Synaptic Systems); BRCA1 (20649-1-AP, Proteintech); H2A.X (A11361, ABclonal); γH2A.X (AP0687, ABclonal ); GAPDH(60004-1-Ig, Proteintech); eIF3A (3411T, CST); Flag (F1804, Sigma-Aldrich); anti‐rabbit IgG HRP‐ linked antibody (7074; CST); Peroxidase-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (SA00001-1, Proteintech); α-Tubulin (66031-1-Ig, Proteintech); CHK1(bs-1681R, Bioss); CHK2 (252092, Zenbio); p-CHK2-S19(AP0862, ABclonal); Anti-HA tag monoclonal antibody (TA100012, OriGene); Mouse-IgG (BA1046, Boster); Rabbit IgG (B900610, Proteintech); p21(10355-1-AP, Proteintech); 53BP1 (BA2878, Boster); CDK1 (D160158, BBI life sciences); CDK2 (D199431, BBI life sciences); Cyclin B2 (21644-1-AP, Proteintech).

Techniques: Modification, Expressing

Journal: Metabolites

Article Title: Lifestyle Intervention Therapy Modulates Global DNA Methylation and Adipogenic Gene Expression in Severely Obese Hypogonadal Men

doi: 10.3390/metabo16030198

Figure Lengend Snippet: Lifestyle intervention with or with aromatase inhibitors (LSI ± AI) alters expression of adipogenic and metabolic genes. mRNA expression levels of PPARγ , CEBPα , and FTO were measured in PBMCs before (BL/baseline) and after the lifestyle intervention (12 M). Bars represent the mean fold change ± SEM. Expression of all three genes significantly decreased following the intervention, indicating reduced adipogenic and metabolic activity associated with LSI ± AI.

Article Snippet: FAM-labeled TaqMan gene expression assays (Applied Biosystem, College Station, TX, USA) were used for PPARγ (Assay ID:), CEBPα (Hs00269972_s1:), FTO (Hs01057145_m1), DNMT1 (Hs00945875_m1), DNMT3A (Hs01027162_m1), and DNMT3B (Hs00171876_m1), and a VIC-labeled TaqMan gene expression assay for housekeeping 18S (assay ID: Hs03928990_g1).

Techniques: Expressing, Activity Assay

Journal: Stem Cell Reports

Article Title: METTL3 uncouples chromatin accessibility from transcription during retinal development

doi: 10.1016/j.stemcr.2025.102690

Figure Lengend Snippet: METTL3 regulates gene expression primarily through m6A-mediated RNA stability (A) Motif analysis of lost m6A sites ( n = 27,832) identified by GLORI in WT vs. Mettl3 KO organoids after 4 days in RPC differentiation conditions reveals m6A-specific DRACH consensus motif; n = 3 independent experiments. (B) Curve graph of m6A modification ratio in WT and Mettl3 KO organoids after 4 days in RPC differentiation conditions; n = 3 independent experiments. (C) Metagene profiles showed the distribution of m6A sites between WT ( n = 28,689) and Mettl3 KO ( n = 11,881) WT and Mettl3 KO organoids after 4 days in RPC differentiation conditions; n = 3 independent experiments. (D) Catalytic mutant Mettl3 (APPA) enhances mRNA stability of up-regulated differentially expressed genes (DEGs) in Mettl3 KO organoids versus Mettl3 WT organoids. RNA stability assay was performed via actinomycin D treatment at day 6 from Mettl3 KO organoids reconstituted with FLAG-METTL3 WT or FLAG-METTL3 APPA lines for 0, 2, 4, 6, and 8 h, and total RNA was subjected to mRNA-seq. (E) FTO-dCas13b-HA expression detected by HA immunoprecipitation followed by HA antibody detection in Rx:GFP mESCs with or without Dox (2 μg/mL) treatment. (F) Fluorescence microscopy images show high infection efficiency in day 6 Rx:GFP + RPCs when infected at day 0 with Lenti-mCherry gRNAs; n = 3 independent experiments. (G–J) Single-base elongation and ligation-based qPCR amplification (SELECT) for the effect of FTO-dCas13b-mediated m6A removal at A1745 site of Six3 3′UTR increased Six3 transcript level; n = 3 independent experiments. In contrast, (I and J) SELECT assay showed that m6A removal at A789 site of Six3 CDS had no effect on Six3 mRNA level; n = 3 independent experiments.

Article Snippet: To investigate m6A modification function at mRNAs such as Six3 3′UTR, we first generated a Dox-inducible, HA-tagged FTO-dCas13b (FTO from Addgene 134781 and dCas13b from Addgene 103865) stable overexpression line in Rx:GFP mESCs.

Techniques: Gene Expression, Modification, Mutagenesis, Stability Assay, Expressing, Immunoprecipitation, Fluorescence, Microscopy, Infection, Ligation, Amplification