Journal: Cell death & disease

Article Title: METTL3-mediated m6A modification of LINC00839 maintains glioma stem cells and radiation resistance by activating Wnt/β-catenin signaling.

doi: 10.1038/s41419-023-05933-7

Figure Lengend Snippet: Fig. 4 YTHDF2 stabilizes LINC00839. A FISH and IF double staining in GSCs shows the co-localization of LINC00839 (Cy3; Red) and YTHDF2 (Green); Nuclei are stained blue (DAPI). Scale bar: 10 μm. B LINC00839 expression in GSCs transfected with control or YTHDF2 shRNA was detected by qRT-PCR. **p < 0.001. C LINC00839 expression in GSCs transfected with vector or YTHDF2 was detected by qRT-PCR. **p < 0.001. D LINC00839 RNA stability in control and YTHDF2-silenced cells. qRT-PCR of LINC00839 at the indicated time points after treatment with actinomycin D (10 μg/mL). E YTHDF2 was immunoprecipitated and RIP-qPCR was used to assess the association of LINC00839 with YTHDF2. **p < 0.001. F Enrichment of LINC00839 in the immunoprecipitated RNA fraction of GSCs following overexpression of either wildtype (WT) YTHDF2 or m6A-binding mutant YTHDF2 (W432A and W486A) in GSCs. **p < 0.001. G LINC00839 expression in GSCs expressing either WT or mutant YTHDF2 was detected by qRT-PCR. **p < 0.001.

Article Snippet: Antibodies against LP0 (ab192866, Abcam); Olig2 (65915, CST); SOX2 (3579, CST); MYC (18583, CST); γ-H2AX (9718, CST); WTAP (41934, CST); METTL3 (86132, CST); YTHDF2 (71283, CST); YTHDF1 (43123, CST); YTHDF3 (24206, CST); YTHDC1 (81504, CST); YTHDC2 (46324, CST); EIF3 (3411, CST); hnRNPA2B1 (9304, CST); β-catenin (8480, CST); Flag (SAB4301135, Sigma); c-Src (2109, CST); β-catenin pY654 (ab59430, Abcam); β-catenin pY333 (ab119363, Abcam); E-cadherin (14472, CST); α-Tubulin (3873, CST) were used for western blotting assays.

Techniques: Double Staining, Staining, Expressing, Transfection, Control, shRNA, Quantitative RT-PCR, Plasmid Preparation, Immunoprecipitation, Over Expression, Binding Assay, Mutagenesis

Journal: Cell death & disease

Article Title: METTL3-mediated m6A modification of LINC00839 maintains glioma stem cells and radiation resistance by activating Wnt/β-catenin signaling.

doi: 10.1038/s41419-023-05933-7

Figure Lengend Snippet: Fig. 6 LINC00839 promotes c-Src-mediated β-catenin phosphorylation. A The enrichment of c-Src in pull-down products of LINC00839 was measured by western blot. B RNA immunoprecipitation with an anti-c-Src antibody was used to assess whether c-Src binding to LINC00839 in GSCs. C GSCs were transfected with or without RNase If. Immunoprecipitation with anti-β-catenin and anti-c-Src antibodies were performed. D GSCs were transfected with or without RNase If. IF assays were performed to analyze the co-localization of β-catenin (Red) and c-Src (Green). E GSCs were transfected with control or LINC00839 ASO. Immunoprecipitation with an anti-β-catenin antibody was performed. F Control or METTL3 deleted GSCs were co-transfected with LINC00839. Immunoprecipitation with an anti-β-catenin antibody was performed. G Control or YTHDF2 deleted GSCs were co-transfected with LINC00839. Immunoprecipitation with an anti-β-catenin antibody was performed. H. GSCs were transfected with FL or TL LINC00839. Immunoprecipitation with an anti-β-catenin antibody was performed. I In vitro β-catenin phosphorylation assay using recombinant β-catenin, c-Src proteins, and in vitro-transcribed RNA transcripts as indicated in NETN buffer with the presence of 500 µM ATP. Immunoblots were used to detect β-catenin phosphorylation level. J Luciferase activity (TOP/FOP) in control or LINC00839 overexpressing MES28 co-transfected with WT or Y654E mutation β-catenin. **p < 0.001. K Control or LINC00839 overexpressing GSCs were co-transfected with WT or Y654E mutation β-catenin. Representative images of spheres were photographed. Scale bars, 1 mm. L, M Control or LINC00839 overexpressing GSCs were co-transfected with WT or Y654E mutation β-catenin. The apoptotic rates were measured by flow cytometry. **p < 0.001.

Article Snippet: Antibodies against LP0 (ab192866, Abcam); Olig2 (65915, CST); SOX2 (3579, CST); MYC (18583, CST); γ-H2AX (9718, CST); WTAP (41934, CST); METTL3 (86132, CST); YTHDF2 (71283, CST); YTHDF1 (43123, CST); YTHDF3 (24206, CST); YTHDC1 (81504, CST); YTHDC2 (46324, CST); EIF3 (3411, CST); hnRNPA2B1 (9304, CST); β-catenin (8480, CST); Flag (SAB4301135, Sigma); c-Src (2109, CST); β-catenin pY654 (ab59430, Abcam); β-catenin pY333 (ab119363, Abcam); E-cadherin (14472, CST); α-Tubulin (3873, CST) were used for western blotting assays.

Techniques: Phospho-proteomics, Western Blot, RNA Immunoprecipitation, Binding Assay, Transfection, Immunoprecipitation, Control, In Vitro, Recombinant, Luciferase, Activity Assay, Mutagenesis, Cytometry

Journal: BMC Cancer

Article Title: N6-methyladenosine RNA modification (m6A) is of prognostic value in HPV-dependent vulvar squamous cell carcinoma

doi: 10.1186/s12885-022-10010-x

Figure Lengend Snippet: Summary of the analyzed m6A proteins as indicated and their correlation with overall survival (indicated as %alive) for the entire study cohort, HPV-independent, and HPV-dependent VSCC. The HPV-status was not available for 24 patients. Samples were grouped according to high and low expression based on the staining intensities. p -values for the group comparisons are based on log-rank tests (significance threshold p < 0.5). q -values are based on multiple hypotheses testing using the method of Benjamini and Hochberg with a significance threshold of q < 0.1

Article Snippet: Immunostaining of METTL3, METTL4, METTL14, WTAP, KIAA1429, FTO, ALKBH5, HNRNPA2B1, HNRNPC, YTHDC1, YTHDF1,YTHDF2, and YTHDF3 was performed on the TMAs using an automated staining system (BenchMark ULTRA; Ventana Medical Systems) which performed deparaffinization, pretreatment with cell conditioning buffer (CC1 buffer, pH8), and incubation with primary antibodies (FTO (1:50; Atlas Antibodies #HPA041086), ALKBH5 (1:200; Novus #NBP1-82,188), METTL3 (1:1000; Biorbyt #orb374082), METTL4 (1:40; Atlas Antibodies #HPA040061), METTL14 (1:100; Atlas Antibodies #HPA038002), WTAP (1:100; Atlas Antibodies #HPA010550), KIAA1429 (1:25; Atlas Antibodies #HPA031530), HNRNPC (1:25; Atlas Antibodies #HPA051075), HNRNPA2B1 (1:100; Atlas Antibodies #HPA001666), YTHDC1 (1:25; Atlas Antibodies #HPA036462), YTHDF1 (1:10; Biorbyt #orb179018), YTHDF2 (1:200; Biorbyt #orb39199), YTHDF3 (1:200; Biorbyt #orb374095) at 4 °C overnight.

Techniques: Expressing, Staining

Journal: Nature Communications

Article Title: Fat mass and obesity-associated protein regulates RNA methylation associated with depression-like behavior in mice

doi: 10.1038/s41467-021-27044-7

Figure Lengend Snippet: a Schematics of the strategy used to identify the targets. b Overlap of the genes that are hypermethylated in KD and cKO mice and hypomethylated in OE mice. c Thirty-four genes that are hypermethylated in KD and cKO mice and hypomethylated in OE mice. d – l m 6 A, mRNA, and protein levels of Adrb2 in the hippocampus of KD ( d – f ) cKO ( g – i ) and OE ( j – l ) mice. d n = 6 per group, p = 0.0087; e n = 8 per group, p = 0.0381; f EGFP, n = 3; KD, n = 4, p = 0.0243; g EGFP, n = 5; cKO, n = 6, p = 0.0012; h EGFP, n = 7; cKO, n = 6, p = 0.0269; i n = 3 per group, p = 0.0053; j n = 8 per group, p = 0.0032 k n = 8 per group, p = 0.0022; l n = 3 per group, p = 0.0232. The detection of ADRB2 in panel l was in the same experiment as the detection of SIRT1 in OE in panel e of Fig. . m Expression of Adrb2 mRNA in the hippocampus of MDD patients and healthy controls. n = 3 per group, p = 0.0115. n , o Adrb2 mRNA stability assay after transfection of Neuro-2a cells with plasmids expressing Fto -shRNA or Fto . n = 4 per time point. n Ctl-shRNA vs. KD, 6 h, p = 0.0053; o EGFP vs. OE, 6 h, p = 0.0383. p Levels of Ythdf2 and Adrb2 mRNA after knockdown of Ythdf2 in Neuro-2a cells. n = 4 per group. Ythdf2 , p = 0.0130003; Adrb2 , p = 0.000153. q , r Relative luciferase activity of pMIR-GLO- Adrb2 -3′UTR, with either wild-type (WT Adrb2 3' UTR fragement) or mutant m 6 A sites (Mutant Adrb2 3' UTR fragement) after co-transfection with control vector (Ctl), FTO (FTO), or FTO-mutant (FTO-Mut) into HEK293T cells for 48 h. Firefly luciferase activity was measured and normalized to Renilla luciferase activity, n = 4 per group. q Ctl vs. FTO, p = 0.0002; FTO vs. FTO-Mut, p = 0.0002 r Ctl vs. FTO, p = 0.5376; FTO vs. FTO-Mut, p = 0.1666. * p < 0.05, ** p < 0.01, *** p < 0.001. Two-tailed Student’s t -test for d – m & p ; two-way ANOVA with repeated measures followed by post hoc Tukey’s test for n & o . Adjustments were made for multiple comparisons test. Error bars represent s.e.m. Source data are provided as a Source Data file.

Article Snippet: Plasmids were transfected into Neuro-2a cells using Neofect™ DNA transfection reagent (TF201201, China). siRNA targeting Ythdf2 (sc-155424, Santa Cruz) was transfected into Neuro-2a cells using RNAi MAX (13778150, Thermo).

Techniques: Expressing, Stability Assay, Transfection, shRNA, Luciferase, Activity Assay, Mutagenesis, Cotransfection, Plasmid Preparation, Two Tailed Test

Journal: iScience

Article Title: Epitranscriptomic reader YTHDF2 regulates SEK1( MAP2K4 )-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress

doi: 10.1016/j.isci.2024.110619

Figure Lengend Snippet: Mn treatment decreases YTHDF2 in cell culture (A) Primary mouse astrocytes were exposed to 100 μM for 1–24 h and YTHDF2 levels were determined by Western blot. The bottom panel shows the results of densitometric analysis of YTHDF2 bands normalized by β-actin (n = 4–5). YTHDF2 decreases time-dependently beginning after 3 h in primary mouse astrocytes. (B) ICC representation at 40x depicting YTHDF2 decreases in Mn-exposed primary mouse astrocytes at 24 h (100 μm scale). (C) 24-h treatment of human U251 astrocytes with different heavy metals, all at 100 μM ( n = 3). (D) Mn decreases YTHDF2 mRNA at 24 h in U251 astrocytes ( n = 5). (E) Mn decreases global m6A levels at 24 h in U251 astrocytes, as measured by LC-MS/MS ( n = 6). Data are means ± SEM. Two-group comparisons performed using unpaired t-test. p -values < 0.05 considered significant evidence.

Article Snippet: YTHDF2 pLenti-C-mGFP-P2A-Puro , OriGene , Cat# RC230306L4.

Techniques: Cell Culture, Western Blot, Liquid Chromatography with Mass Spectroscopy

Journal: iScience

Article Title: Epitranscriptomic reader YTHDF2 regulates SEK1( MAP2K4 )-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress

doi: 10.1016/j.isci.2024.110619

Figure Lengend Snippet: YTHDF2 levels can affect pro-inflammatory chemokine/cytokine responses in Mn-exposed astrocytes (A) Validation of YTHDF2 knockdown using siRNA, both qPCR and immunoblotting (n = 5–6). (B) Validation of YTHDF2 overexpression, both qPCR and immunoblotting ( n = 3). (C) siYTHDF2 increased basal pro-inflammatory gene expression and exacerbated after Mn exposure ( n = 3). (D) Overexpression of YTHDF2 suppressed basal pro-inflammatory gene expression and prevented upregulation after Mn exposure ( n = 4). (E and F) Multiplex ELISA analysis of the treatment media of siYTHDF2 and YTHDF2 overexpression experiments for cytokines/chemokines (n = 4–6). IL-8 was most consistently affected by YTHDF2 levels, showing exacerbated release in siYTHDF2 experiments, while its release was prevented in YTHDF2 overexpression experiments. MCP1 (MCAF), IFNγ, IL-6, and MIP1b showed similar trends but with less consistency overall. Data are means ± SEM. Two-group comparisons performed using unpaired t-test. p -values < 0.05 considered significant evidence. Two-way ANOVA with FDR Two-stage step-up method of Benjamini, Krieger, and Yekutieli for multi-group comparison. Q-values < 0.05 considered significant evidence.

Article Snippet: YTHDF2 pLenti-C-mGFP-P2A-Puro , OriGene , Cat# RC230306L4.

Techniques: Knockdown, Western Blot, Over Expression, Expressing, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: iScience

Article Title: Epitranscriptomic reader YTHDF2 regulates SEK1( MAP2K4 )-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress

doi: 10.1016/j.isci.2024.110619

Figure Lengend Snippet: RNA- and RIP-sequencing of Mn-exposed U251 astrocytes reveals YTHDF2 targeting of MAP2K4 (SEK1) (A and B) (A) Z-scores of Mn/Ctrl GO for Transcriptional Factor Targets, indicating important roles for cJUN and HIF1α (B) RIP-sequencing graphical representation of statistically significant YTHDF2 targets in Ctrl that were affected by Mn exposure. YTHDF2 targets are identified as having ≥+1 log2(RIP/input) ratio. MAP2K4 was identified as a lost YTHDF2 target under Mn exposure, suggesting regulation of the SEK1( MAP2K4 )-JNK-cJUN pathway. (C) si YTHDF2 astrocytes present with longer MAP2K4 mRNA half-life ( n = 3). (D) ICC representation at 40x depicting si YTHDF2 astrocytes have increased SEK1 protein levels (100 μm scale). (E) YTHDF2 overexpressing astrocytes present with shorter MAP2K4 half-life under Mn exposure (n = 4–5). (F) ICC representation at 40x depicting YTHDF2-overexpressing astrocytes have decreased SEK1 protein levels (100 μm scale). (G and H) Immunoblotting and quantification revealing cJUN phosphorylation is increased in Mn-exposed astrocytes and sustained in siYTHDF2 Mn-exposed astrocytes. SEK1 protein levels are basally increased in siYTHDF2 astrocytes (n = 3–4). (I and J) Immunoblotting and quantification revealing cJUN phosphorylation is increased in Mn-exposed astrocytes and prevented in YTHDF2-overexpressing Mn-exposed astrocytes. SEK1 protein levels are basally decreased in YTHDF2-overexpressing astrocytes ( n = 4). Data are means ± SEM. Two-group comparisons performed using unpaired t-test, with 2-fold gene threshold and 1.96 Z - score threshold using Altanalyze. Adjusted p -values < 0.05 considered significant evidence. For mRNA half-life comparisons, One-phase decay non-linear regression analysis was performed. p -values < 0.05 considered significant evidence. two-way ANOVA with FDR Two-stage step-up method of Benjamini, Krieger, and Yekutieli for multi-group comparison. Q-values < 0.05 considered significant evidence.

Article Snippet: YTHDF2 pLenti-C-mGFP-P2A-Puro , OriGene , Cat# RC230306L4.

Techniques: Sequencing, Western Blot, Comparison

Journal: iScience

Article Title: Epitranscriptomic reader YTHDF2 regulates SEK1( MAP2K4 )-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress

doi: 10.1016/j.isci.2024.110619

Figure Lengend Snippet: YTHDF2 is decreased in Mn-gavaged mice, and a conditional knockout of YTHDF2 in the astrocytes of mice leads to increased astrocyte reactivity in substantia nigra pars compacta/reticulata and globus pallidus (A) IHC representation at 40x depicting YTHDF2 decreases and colocalization in GFAP+ cells in the globus pallidus of Mn-gavaged mice (100 μm scale). (B) Immunoblotting of the substantia nigra showing decreases of YTHDF2 in mice gavaged with Mn ( n = 9). (C) Schematic of Y2cKO mice generation. Tamoxifen (tiY2cKO) induces Cre recombination of YTHDF2 exon 4 to produce a band at 748 bp in astrocytes isolated from striatal/hippocampal brain tissue using biotinylated-EAAT1/GLAST-1 antibody. (D) Globus pallidus validation of YTHDF2 protein reduction and increased GFAP immunoreactivity, and also increased m6A staining upon YTHDF2 deletion. YTHDF2 was quantified by parent-child extraction using GFAP as the parent (n = 3–4) (100 μm scale). (E) Globus pallidus representative images of increased C3d immunoreactivity in GFAP-positive cells in the tiY2cKO mice (100 μm scale). (F) Immunoblotting and quantification of substantia nigra- showing tiY2cKO mice with increased GFAP immunoreactivity similar to Mn-gavaged mice, indicating specific loss of YTHDF2 in astrocytes increases their reactivity (n = 4–6). (G) Substantia nigra representative images of increased C3d immunoreactivity in GFAP-positive cells in the tiY2cKO mice (100 μm scale). Data are means ± SEM. Student’s t test or two-way ANOVA with FDR Two-stage step-up method of Benjamini, Krieger, and Yekutieli for multi-group comparison. Q-values < 0.05 considered significant evidence, with q-values between 0.05 and 0.01 considered as weaker evidence taking into consideration variations in data and trends.

Article Snippet: YTHDF2 pLenti-C-mGFP-P2A-Puro , OriGene , Cat# RC230306L4.

Techniques: Knock-Out, Western Blot, Isolation, Staining, Extraction, Comparison

Journal: iScience

Article Title: Epitranscriptomic reader YTHDF2 regulates SEK1( MAP2K4 )-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress

doi: 10.1016/j.isci.2024.110619

Figure Lengend Snippet: Integrated working hypothesis of YTHDF2’s role on the SEK1( MAP2K4 )-JNK-cJUN pathway Upon Mn exposure, ALKBH5 levels increase while YTHDF2 decreases, leading to increased half-life of MAP2K4 mRNA. Increased abundance of MAP2K4 leads to more SEK1 protein, allowing for the sustained activation (increased phosphorylation) of the downstream pathway targets JNK and cJUN by Mn, which promotes the pro-inflammatory response in astrocytes. When YTHDF2 is overexpressed, MAP2K4 mRNA is degraded. This leads to lower SEK1 protein levels, reducing the pathway activation of JNK and cJUN (decreased phosphorylation) by Mn, resulting in an anti-inflammatory response.

Article Snippet: YTHDF2 pLenti-C-mGFP-P2A-Puro , OriGene , Cat# RC230306L4.

Techniques: Activation Assay

Journal: iScience

Article Title: Epitranscriptomic reader YTHDF2 regulates SEK1( MAP2K4 )-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress

doi: 10.1016/j.isci.2024.110619

Figure Lengend Snippet:

Article Snippet: YTHDF2 pLenti-C-mGFP-P2A-Puro , OriGene , Cat# RC230306L4.

Techniques: Recombinant, Extraction, Selection, Negative Control, Software