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: YTHDF2 governs muscle size through a targeted modulation of proteostasis.

doi: 10.1038/s41467-024-46546-8

Figure Lengend Snippet: Fig. 4 | YTHDF2-null muscles favor catabolic signaling. a Western blot analyses and quantification using total protein detection by Ponceau stain as a loading control for b phosphor-(p-)AKT, c AKT, d p-AKT/AKT ratio, e p-mTOR, f mTOR, g p-mTOR/AKT ratio, h LC3I, i LC3II, j LC3II/LC3I ratio, k p62, l Beclin-1, m monoubiquitin, and n polyubiquitin in Ctrl and Y2-KO quadriceps at 8 months of age. o Proteasomal activity as determined via colorimetric proteasome activity

Article Snippet: Immunoprecipitation was achieved by incubating 3mg of protein extract with 5 μg YTHDF2 antibody (Proteintech, #24744-1-AP) or control anti-rabbit IgG (Millipore Sigma, #12-370) at 4 °C overnight.

Techniques: Muscles, Western Blot, Staining, Control, Activity Assay

Journal: Journal of Ovarian Research

Article Title: YTHDF2, a protein repressed by miR-145, regulates proliferation, apoptosis, and migration in ovarian cancer cells

doi: 10.1186/s13048-020-00717-5

Figure Lengend Snippet: The expression of YTHDF2 in ovarian cancer tissues. a Relative expression of YTHDF2 in ovarian cancer tissues and normal ovarian tissues. b The relationship between the expression level of YTHDF2 and clinical stage. c The relationship between the expression level of YTHDF2 and pathological grade. d The relationship between the expression level of YTHDF2 and metastasis. All experiments were carried out in triplicate and the results were presented as means ± SE. * P < 0.05, ** P < 0.001, t-test. N normal ovarian tissue, C ovarian cancer tissues

Article Snippet: The human YTHDF2 expression vector pcDNA3-flag-YTHDF2 were obtained from Addgene (Boston, MA, USA).

Techniques: Expressing

Journal: Journal of Ovarian Research

Article Title: YTHDF2, a protein repressed by miR-145, regulates proliferation, apoptosis, and migration in ovarian cancer cells

doi: 10.1186/s13048-020-00717-5

Figure Lengend Snippet: YTHDF2 significantly downregulates global mRNA m6A levels to promotes EOC cell proliferation and migration. a The mRNA and protein level of YTHDF2 was downregulated after transfection of YTHDF2 siRNA. b Knocking down YTHDF2 increased global mRNA m6A level. (C)CCK8 assay result showed knock-down of YTHDF2 decreased proliferation of ovarian cancer cells. d Knock-down of YTHDF2 promoted apoptosis of ovarian cancer cells. e Knock-down of YTHDF2 inhibited migration of ovarian cancer cells. f The mRNA and protein level of YTHDF2 was upregulated after overexpression of YTHDF2. g overexpression of YTHDF2 decreased global mRNA m6A level. (H)CCK8 assay result showed upregulated of YTHDF2 increased proliferation of ovarian cancer cells. (I) overexpression of YTHDF2 inhibited apoptosis of ovarian cancer cells. j overexpression of YTHDF2 promoted migration of ovarian cancer cells. All experiments were carried out in triplicate and the results were presented as means ± SE. * P < 0.05, ** P < 0.001, t-test

Article Snippet: The human YTHDF2 expression vector pcDNA3-flag-YTHDF2 were obtained from Addgene (Boston, MA, USA).

Techniques: Migration, Transfection, CCK-8 Assay, Over Expression

Journal: Journal of Ovarian Research

Article Title: YTHDF2, a protein repressed by miR-145, regulates proliferation, apoptosis, and migration in ovarian cancer cells

doi: 10.1186/s13048-020-00717-5

Figure Lengend Snippet: YTHDF2 is the direct target gene of miR-145. a Scatter diagram showing. YTHDF2 expression and miR-145 expression by qRT-PCR and their correlations (R 2 = 0.914, P = 0.000) in 31 EOC tissue samples. b qRT-PCR results showed a negative correlation between YTHDF2 and miR-145 in both SKOV3 and 3AO cells. c qRT-PCR showed that transfection of miR-145 mimic rescued miR-145 level in SKOV3 and 3AO cells. d The mRNA and protein levels of YTHDF2 decreased after overexpression of miR-145. e The expression level of miR-145 increased after overexpression of YTHDF2. f Luciferase reporter assays showed that miR-145 targeted YTHDF2 directly. All experiments were carried out in triplicate and the results were presented as means ± SE. *P < 0.05, **P < 0.001, t-test

Article Snippet: The human YTHDF2 expression vector pcDNA3-flag-YTHDF2 were obtained from Addgene (Boston, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Over Expression, Luciferase

Journal: Journal of Ovarian Research

Article Title: YTHDF2, a protein repressed by miR-145, regulates proliferation, apoptosis, and migration in ovarian cancer cells

doi: 10.1186/s13048-020-00717-5

Figure Lengend Snippet: Overexpression of YTHDF2 rescues miR-145-induced reduction of proliferation and migration in EOC. a overexpression of YTHDF2 reversed the proliferation repressed by overexpression of miR-145. b miR-145 promoted apoptosis of ovarian cancer cells, which was counteracted by overexpression of YTHDF2. c overexpression of YTHDF2 reversed the migration repressed by overexpression of miR-145. d The effect of overexpression of miR-145 on the global mRNA m6A levels was offset by overexpression of YTHDF2. e The expression of YTDHF2 at mRNA levels after overexpression of miR-145 and YTHDF2. f The expression of YTDHF2 at protein levels after overexpression of miR-145 and YTHDF2. All experiments were carried out in triplicate and the results were presented as means ± SE. * P < 0.05, ** P < 0.001, t-test

Article Snippet: The human YTHDF2 expression vector pcDNA3-flag-YTHDF2 were obtained from Addgene (Boston, MA, USA).

Techniques: Over Expression, Migration, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Epitranscriptional regulation of TGF- β pseudoreceptor BAMBI by m 6 A/YTHDF2 drives extrinsic radioresistance

doi: 10.1172/JCI172919

Figure Lengend Snippet: ( A ) Integrative Genomics Viewer tracks displaying the distribution of m 6 A peaks and YTHDF2-binding peaks across Bambi transcripts, based on the MeRIP-Seq and RIP-Seq of MC38 tumor–infiltrating myeloid cells. ( B ) Graphs showing enrichment of Bambi mRNA in the YTHDF2-immunoprecipitated RNA fraction of BM-MDSCs, determined by RIP-qPCR. Rabbit IgG served as a control. Enrichment of the indicated genes was normalized to the input level. n = 3 per group. ( C ) MDSCs were sorted from BM-derived cells from WT and Ythdf2 -cKO ( Lyz2 cre Ythdf2 fl/fl ) mice; WT MDSCs were directly treated with IR (4 Gy and cultured for 6 hours) (WT+IR). MDSCs were treated with actinomycin D. mRNA was collected at indicated time points after treatment and mRNA levels of Bambi were measured by RT-qPCR. n = 3 per group. ( D ) qPCR analysis of Bambi mRNA levels in different MDSCs isolated from MC38 tumors in WT, WT+IR, Ythdf2 -cKO, and Ythdf2 -cKO+IR mice 3 days after IR. n = 5 per group. ( E ) MFI of BAMBI in MC38 tumor–infiltrating MDSCs (as indicated) by flow cytometry analysis 3 days after IR. n = 6 per group. ( F ) WT-YTHDF2 and m 6 A-binding site-mutated YTHDF2-overexpressing Ythdf2 -deficient CD45.2-BM-MDSCs ( Ythdf2 -cKO+WT, Ythdf2 -cKO+Mut, respectively) were obtained via lentiviral transfection. The BM-MDSCs were used for adoptive transfer into MC38 tumor–bearing CD45.1 mice. On the same day, mice were treated with local tumor irradiation. Three days after IR, tumors were harvested to measure the MFI of BAMBI in newly infiltrated CD45.2-MDSCs by flow cytometry. n = 5 per group. Data are represented as means ± SEM. One of 2 or 3 representative experiments is shown ( B – F ). Statistical analysis was performed using 2-sided, unpaired Student’s t test ( B ) or 1-way ANOVA with Bonferroni’s multiple-comparison tests ( C – F ). * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: RIP for YTHDF2 was performed using 10 to 20 μg anti-YTHDF2 rabbit polyclonal antibody (catalog ARP67917_P050; Aviva Systems Biology).

Techniques: Binding Assay, Immunoprecipitation, Control, Derivative Assay, Cell Culture, Quantitative RT-PCR, Isolation, Flow Cytometry, Transfection, Adoptive Transfer Assay, Irradiation, Comparison