Journal: Microorganisms

Article Title: Coxsackievirus B3-Induced m 6 A Modification of RNA Enhances Viral Replication via Suppression of YTHDF-Mediated Stress Granule Formation

doi: 10.3390/microorganisms12112152

Figure Lengend Snippet: CVB3 infection induces subcellular redistribution of METTL3, ALKBH5 and YTHDF2. ( A ) METTL3 and ( B ) ALKBH5 translocate from the nucleus to the cytoplasm. HeLa cells were infected with CVB3 (10 MOI) or sham-infected with PBS for 5 h. Confocal images show immunofluorescent staining results. The nucleus (blue) was stained with DAPI. Viral protein VP1 (pink) and METTL3 or ALKBH5 (green) were stained using specific antibodies. ( C ) m 6 A reader protein YTHDF2 translocates from the cytoplasm to the nucleus. Cells were treated as described above but stained with an anti-YTHDF2 antibody. Note that at 5 h post infection (hpi), some VP1 also translocated to the nucleus.

Article Snippet: Polyclonal rabbit anti-human YTHDF1 (66745-1AP), YTHDF2 (24744-1AP), YTHDF3 (25537-1-AP), METTL3 (15073-AP) and METTL14 (23158-1-AP) (Proteintech, Rosemont, IL, USA); polyclonal rabbit anti-human ALKBH5 (Abcam, Cambridge, United Kingdom); monoclonal mouse anti-Flag tag (Sigma) and monoclonal mouse anti-3D antibody (GeneTex, Irvine, CA, USA).

Techniques: Infection, Staining

Journal: Microorganisms

Article Title: Coxsackievirus B3-Induced m 6 A Modification of RNA Enhances Viral Replication via Suppression of YTHDF-Mediated Stress Granule Formation

doi: 10.3390/microorganisms12112152

Figure Lengend Snippet: CVB3 infection induces cleavage of reader proteins YTHDF1-3 and increases CVB3 VP1 production and 2A transcription. HeLa cells were infected with CVB3 or sham-infected with PBS. Cell lysates were collected at 3 and 5 hpi for Western blot analysis to detect YTHDF2 ( A ), YTHDF1 ( B ), and YTHDF3 ( C ), as well as viral VP1. ( D ) HeLa cells were transfected with YTHDF2 siRNA (siY2) or control siRNA (siCtrl) and then infected with CVB3. ( G ) HeLa cells were transfected with a YTHDF2 plasmid (pY2) or an empty vector and then infected with CVB3. Western blot was conducted using the indicated antibodies ( D , G ), and protein levels were quantified using the ImageJ program ( E , H ). RT-qPCR was conducted using samples from ( D , G ) to measure the transcripts of the viral gene 2A ( F , I ). Data are presented as means ± SEM, n = 3, * p < 0.05, ** p < 0.01.

Article Snippet: Polyclonal rabbit anti-human YTHDF1 (66745-1AP), YTHDF2 (24744-1AP), YTHDF3 (25537-1-AP), METTL3 (15073-AP) and METTL14 (23158-1-AP) (Proteintech, Rosemont, IL, USA); polyclonal rabbit anti-human ALKBH5 (Abcam, Cambridge, United Kingdom); monoclonal mouse anti-Flag tag (Sigma) and monoclonal mouse anti-3D antibody (GeneTex, Irvine, CA, USA).

Techniques: Infection, Western Blot, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR

Journal: Microorganisms

Article Title: Coxsackievirus B3-Induced m 6 A Modification of RNA Enhances Viral Replication via Suppression of YTHDF-Mediated Stress Granule Formation

doi: 10.3390/microorganisms12112152

Figure Lengend Snippet: Colocalization and interaction of YTHDF2 with HuR in SGs. ( A ) HeLa cells were either infected with CVB3 (MOI 10) or sham-infected with PBS. The cells were fixed at 3 and 5 hpi and then subjected to immunofluorescent staining for YTHDF2, HuR and DAPI. Images were captured by confocal microscopy. Red indicates YTHDF2, green indicates HuR and blue indicates DAPI. Scale bar: 10 µm. ( B ) The number of SGs per cell was quantified using a total of 25 cells from 5 random microscopic views (n = 25). Data were analyzed by Student’s t -test with Welch’s correction and are presented as means ± SEM, **** p < 0.0001. ( C ) HeLa cells were infected with CVB3 (MOI 10) for 3 h, and the collected cell lysates were used for co-immunoprecipitation (IP) with HuR antibody. The pulled HuR-protein complexes were detected by Western blot using a YTHDF2 antibody. Conversely, cell lysates were also incubated with the YTHDF2 antibody to pull down the complexes and then detected using the HuR antibody. IgG was used as a control.

Article Snippet: Polyclonal rabbit anti-human YTHDF1 (66745-1AP), YTHDF2 (24744-1AP), YTHDF3 (25537-1-AP), METTL3 (15073-AP) and METTL14 (23158-1-AP) (Proteintech, Rosemont, IL, USA); polyclonal rabbit anti-human ALKBH5 (Abcam, Cambridge, United Kingdom); monoclonal mouse anti-Flag tag (Sigma) and monoclonal mouse anti-3D antibody (GeneTex, Irvine, CA, USA).

Techniques: Infection, Staining, Confocal Microscopy, Immunoprecipitation, Western Blot, Incubation, Control

Journal: Microorganisms

Article Title: Coxsackievirus B3-Induced m 6 A Modification of RNA Enhances Viral Replication via Suppression of YTHDF-Mediated Stress Granule Formation

doi: 10.3390/microorganisms12112152

Figure Lengend Snippet: Silencing m 6 A machinery suppresses SG formation. HeLa cells were transfected with specific siRNAs to knock down either METTL3 ( A ) or YTHDF2 ( C ) and then infected with CVB3 at an MOI of 10. Cells were fixed at 3 hpi and then subjected to immunofluorescent staining for HuR and DAPI. Images were captured by confocal microscopy. Green indicates HuR and blue indicates DAPI. Scale bar: 10 µm. ( B , D ) The number of SGs per cell was quantified using a total of 25 cells from 5 random microscopic views (n = 25). Data were analyzed by Student’s t -test with Welch’s correction and are presented as means ± SEM, **** p < 0.0001.

Article Snippet: Polyclonal rabbit anti-human YTHDF1 (66745-1AP), YTHDF2 (24744-1AP), YTHDF3 (25537-1-AP), METTL3 (15073-AP) and METTL14 (23158-1-AP) (Proteintech, Rosemont, IL, USA); polyclonal rabbit anti-human ALKBH5 (Abcam, Cambridge, United Kingdom); monoclonal mouse anti-Flag tag (Sigma) and monoclonal mouse anti-3D antibody (GeneTex, Irvine, CA, USA).

Techniques: Transfection, Knockdown, Infection, Staining, Confocal Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Ythdf2 facilitates precursor miR-378/miR-378-5p maturation to support myogenic differentiation

doi: 10.1007/s00018-024-05456-0

Figure Lengend Snippet: miRNA-seq analysis of Ythdf2-knockdown GPMs at pre-differentiation A GPMs were transfected with either siCtrl or siYthdf2 for 24 h and then induced to undergo myogenic differentiation for 3 days. Cell samples were harvested at day 0 (GM) and day 3 (DM) of differentiation. B The mRNA expression level of Ythdf2 in cells were detected by qRT-PCR. C Phase contrast microscopy images of GPMs at day 0 (GM) and day 3 (D3) of differentiation. D Representative images of immunofluorescence blot for Ythdf2 and MyHC protein in differentiated siCtrl and siYthdf2 cells. Scale bars represent 100 μm. E A volcano plot shows DEMs between the siCtrl_GM and siYthdf2_GM group. F Heatmap shows hierarchical clustering of DEMs in the siCtrl_GM and siYthdf2_GM group. GO enrichment analysis G and KEGG enrichment analysis H of predicted target genes for the DEMs

Article Snippet: Subsequently, the cells were blocked with 3% bovine serum albumin and incubated overnight at 4 °C with the specific antibodies, including Anti- YTHDF2 antibody (1:500 dilution; Proteintech, 24,744–1-AP, Wuhan, China) and Anti-Slow Skeletal myosin heavy chain antibody (1:1000 dilution; Abcam, ab11083, Cambridge, UK).

Techniques: Knockdown, Transfection, Expressing, Quantitative RT-PCR, Microscopy, Immunofluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Ythdf2 facilitates precursor miR-378/miR-378-5p maturation to support myogenic differentiation

doi: 10.1007/s00018-024-05456-0

Figure Lengend Snippet: miRNA-seq analysis of differentiated Ythdf2-knockdown GPMs. GPMs were transfected with either siCtrl or siYthdf2 for 24 h and induced to undergo myogenic differentiation for 3 days before harvest. A A volcano plot shows DEMs between the siCtrl_DM group and siYthdf2_DM group. B Heatmap shows hierarchical clustering of DEMs in the siCtrl_DM group and siYthdf2_DM group. GO enrichment analysis C and KEGG enrichment analysis D of predicted target genes for the DEMs

Article Snippet: Subsequently, the cells were blocked with 3% bovine serum albumin and incubated overnight at 4 °C with the specific antibodies, including Anti- YTHDF2 antibody (1:500 dilution; Proteintech, 24,744–1-AP, Wuhan, China) and Anti-Slow Skeletal myosin heavy chain antibody (1:1000 dilution; Abcam, ab11083, Cambridge, UK).

Techniques: Knockdown, Transfection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Ythdf2 facilitates precursor miR-378/miR-378-5p maturation to support myogenic differentiation

doi: 10.1007/s00018-024-05456-0

Figure Lengend Snippet: Identification of miRNAs involved in Ythdf2-mediated myogenesis. A Venn diagram depicting the common DEMs in siCtrl and siYthdf2 cells at GM and D3 of differentiation. B Heatmap of common DEMs across the different groups. C miRNA–mRNA network established from the DEMs and DEGs in siCtrl and siYthdf2 cells during the GM phase. D miRNA–mRNA network established from the DEMs and DEGs in siCtrl and siYthdf2 cells during the DM phase. Red represents upregulated miRNAs or mRNAs, while purple denotes downregulated one; Δ represents miRNA, and ○ represents mRNA. E Correlation of expression between Ythdf2 and selected DEMs during myogenic differentiation. F The locations of caprine miR-378 and miR-378-5p within the intron of the PPARGC1β gene. G Conservation analysis of miR-378 and miR-378-5p among different species. H Expression levels of miR-378 and miR-378-5p in longissimus muscle of goat at different ages, determined by qRT-PCR. The values are expressed as mean ± SEM; *p < 0.05, **p < 0.01

Article Snippet: Subsequently, the cells were blocked with 3% bovine serum albumin and incubated overnight at 4 °C with the specific antibodies, including Anti- YTHDF2 antibody (1:500 dilution; Proteintech, 24,744–1-AP, Wuhan, China) and Anti-Slow Skeletal myosin heavy chain antibody (1:1000 dilution; Abcam, ab11083, Cambridge, UK).

Techniques: Expressing, Quantitative RT-PCR

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Ythdf2 facilitates precursor miR-378/miR-378-5p maturation to support myogenic differentiation

doi: 10.1007/s00018-024-05456-0

Figure Lengend Snippet: Ythdf2 regulates the expression of miR-378 and miR-378-5p in an m 6 A-dependent manner. A The interaction between Ythdf2 protein and pre-miR-378/miR-378-5p mRNA was analyzed by RIP assay. B and C GPMs were transfected with siCtrl or siYthdf2, as well as overexpression Ythdf2 plasmid (oeYthdf2) or Pex3. The mRNA levels of Ythdf2, pre-miR-378/miR-378-5p, miR-378, and miR-378-5p were quantified by qRT-PCR. D The stability of pre- miR-378/378-5p mRNA in siCtrl and siYthdf2 cells was assessed by qRT-PCR. E The expression levels of Mettl3, Ythdf2, pre-miR-378/miR-378-5p, miR-378 and miR-378-5p were quantified by qRT-PCR in GPMs transfected with either Pex3, oeMettl3, or both oeMettl3 and siYthdf2. The values are expressed as the mean ± SEM; *p < 0.05, **p < 0.01

Article Snippet: Subsequently, the cells were blocked with 3% bovine serum albumin and incubated overnight at 4 °C with the specific antibodies, including Anti- YTHDF2 antibody (1:500 dilution; Proteintech, 24,744–1-AP, Wuhan, China) and Anti-Slow Skeletal myosin heavy chain antibody (1:1000 dilution; Abcam, ab11083, Cambridge, UK).

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Ythdf2 facilitates precursor miR-378/miR-378-5p maturation to support myogenic differentiation

doi: 10.1007/s00018-024-05456-0

Figure Lengend Snippet: Ythd2 binds to DICER1 and TARBP2 and facilitates pre-miR-378/miR-378-5p processing. The mRNA A and protein B levels of DICER1 and TARBP2 in siCtrl and siYthdf2 cells, as detected using qRT-PCR and western blot. C Statistical results of the western blot analysis of B . D Structure of the docking complex between Ythdf2 and DICER1 proteins. E Structure of the docking complex between Ythdf2 and TARBP2 proteins. F Co-IP of Ythdf2-FLAG with endogenous DICER1 and TARBP2 in GPMs transfected with Ythdf2-FLAG. G and H GPMs were transfected with the following combinations: siCtrl + pGCMV-miR vector (siCtrl + pre-miR-NC), siCtrl + pGCMV-pre-miR-378/miR-378-5p plasmid (siCtrl + pre-miR-378/miR-378-5p), siDICER1 + pGCMV-pre-miR-378/miR-378-5p plasmid (siDICER1 + pre-miR-378/miR-378-5p), siYthdf2 + pGCMV-pre-miR-378/miR-378-5p plasmid (siYthdf2 + pre-miR-378/miR-378-5p), and siDICER1 + siYthdf2 + pGCMV-pre-miR-378/miR-378-5p plasmid (siDICER1 + siYthdf2 + pre-miR-378/miR-378-5p). After 48 h, the expression levels of DICER1, Ythdf2, pre-miR-378/miR-378-5p, miR-378, and miR-378-5p in GPMs were detected using qRT-PCR. The values are expressed as the mean ± SEM; *p < 0.05, **p < 0.01. a–d Means with distinct superscripts within the same row indicate significant differences (p < 0.05)

Article Snippet: Subsequently, the cells were blocked with 3% bovine serum albumin and incubated overnight at 4 °C with the specific antibodies, including Anti- YTHDF2 antibody (1:500 dilution; Proteintech, 24,744–1-AP, Wuhan, China) and Anti-Slow Skeletal myosin heavy chain antibody (1:1000 dilution; Abcam, ab11083, Cambridge, UK).

Techniques: Quantitative RT-PCR, Western Blot, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Ythdf2 facilitates precursor miR-378/miR-378-5p maturation to support myogenic differentiation

doi: 10.1007/s00018-024-05456-0

Figure Lengend Snippet: Overexpression of miR-378 rescues Ythdf2 knockdown-induced inhibition of myogenic differentiation. A – D GPMs were transfected with indicated siRNAs (siCtrl, siYthdf2, and siYthdf2 + miR-378). A Fluorescence micrographs (left) of GPMs transfected with the indicated siRNAs after 3 days of differentiation. Scale bars represent 100 μm. Quantitative analysis of the fusion index (right). B qRT-PCR analysis of the mRNA level of Ythdf2, miR-378, MyHC, MyoD1, and myogenin in the differentiated GPMs with the indicated siRNA transfection. C Western blot analysis of the protein level of Ythdf2, MyHC, MyoD1, and myogenin in GPMs with transfection of indicated siRNAs at day 3 of differentiation. D Statistical results of the western blot analysis of C . E Western blot t analysis of the protein levels of p-mTOR, mTOR, p-AMPK, AMPK, p-AKT, and AKT in GPMs transfected with siCtrl, siYthdf2, and siYthdf2 + miR-378 mimics F Statistical results of the western blot analysis of (E). The values are expressed as the mean ± SEM; *p < 0.05, **p < 0.01

Article Snippet: Subsequently, the cells were blocked with 3% bovine serum albumin and incubated overnight at 4 °C with the specific antibodies, including Anti- YTHDF2 antibody (1:500 dilution; Proteintech, 24,744–1-AP, Wuhan, China) and Anti-Slow Skeletal myosin heavy chain antibody (1:1000 dilution; Abcam, ab11083, Cambridge, UK).

Techniques: Over Expression, Knockdown, Inhibition, Transfection, Fluorescence, Quantitative RT-PCR, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Ythdf2 facilitates precursor miR-378/miR-378-5p maturation to support myogenic differentiation

doi: 10.1007/s00018-024-05456-0

Figure Lengend Snippet: Overexpression of miR-378-5p rescues Ythdf2 knockdown-induced inhibition of myogenic differentiation. A Phase contrast microscopy images of GPMs transfected with the indicated siRNAs after 3 days of differentiation. Scale bars represent 100 μm. B qRT-PCR analysis of the mRNA levels of Ythdf2, miR-378-5p, MyHC, MyoD1, and myogenin in the differentiated GPMs with the indicated siRNA transfection. C Western blot analysis of the protein levels of Ythdf2, MyHC, MyoD1, and myogenin in GPMs transfected with the indicated siRNAs at day 3 of differentiation. D Statistical results of the western blot analysis of C . E Western blot t analysis of the protein levels of p-mTOR, mTOR, p-AMPK, AMPK, p-AKT, and AKT in GPMs transfected with siCtrl, siYthdf2, and siYthdf2 + miR-378-5p mimics. F Statistical results of the western blot analysis of E . The values are expressed as the mean ± SEM; *p < 0.05, **p < 0.01

Article Snippet: Subsequently, the cells were blocked with 3% bovine serum albumin and incubated overnight at 4 °C with the specific antibodies, including Anti- YTHDF2 antibody (1:500 dilution; Proteintech, 24,744–1-AP, Wuhan, China) and Anti-Slow Skeletal myosin heavy chain antibody (1:1000 dilution; Abcam, ab11083, Cambridge, UK).

Techniques: Over Expression, Knockdown, Inhibition, Microscopy, Transfection, Quantitative RT-PCR, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Ythdf2 facilitates precursor miR-378/miR-378-5p maturation to support myogenic differentiation

doi: 10.1007/s00018-024-05456-0

Figure Lengend Snippet: Graphical abstract illustrating the role and underlying mechanism of the Ythdf2-mediated pre-miR-378/miR-378-5p maturation in myogenesis. Ythdf2 collaborates with DICER1 and TARBP2 to facilitate the processing of pre-miR-378/miR-378-5p in an m 6 A-dependent manner, thereby supporting myogenic differentiation through the activation of the mTOR pathway

Article Snippet: Subsequently, the cells were blocked with 3% bovine serum albumin and incubated overnight at 4 °C with the specific antibodies, including Anti- YTHDF2 antibody (1:500 dilution; Proteintech, 24,744–1-AP, Wuhan, China) and Anti-Slow Skeletal myosin heavy chain antibody (1:1000 dilution; Abcam, ab11083, Cambridge, UK).

Techniques: Activation Assay

Journal: Cancer Cell International

Article Title: ALKBH5-mediated m 6 A demethylation of lncRNA PVT1 plays an oncogenic role in osteosarcoma

doi: 10.1186/s12935-020-1105-6

Figure Lengend Snippet: YTHDF2 is crucial for ALKBH5-mediated suppression of PVT1 degradation. a The RIP assays using YTHDF2, YTHDF3 and YTHDC2 antibodies were carried out in MG63 and H2OS cells. b The effect of ALKBH5 knockdown on the interaction between YTHDF2 and PVT1 was measured using anti-YTHDF2 RIP assay in MG63 and H2OS cells. c The effect of ALKBH5 overexpression on the interaction between YTHDF2 and PVT1 was measured using anti-YTHDF2 RIP assay in 143B cells. d The YTHDF2 expression was knocked down in ALKBH5-overexpressed 143B cells. e The PVT1 level in ALKBH5-overexpressed 143B cells transfected with YTHDF2 shRNA. f The stability of PVT1 over time in ALKBH5-overexpressed 143B cells transfected with YTHDF2 shRNA was measured by qRT-PCR relative to time 0. *p < 0.05, ***p < 0.001

Article Snippet: Specific antibodies used are listed below: METTL3 (Cell Signaling Technology), YTHDF2 (Cell Signaling Technology) and GAPDH (Proteintech).

Techniques: Knockdown, Over Expression, Expressing, Transfection, shRNA, Quantitative RT-PCR

Journal: Oncoimmunology

Article Title: YTHDF1 and YTHDF2 are associated with better patient survival and an inflamed tumor-immune microenvironment in non–small-cell lung cancer

doi: 10.1080/2162402X.2021.1962656

Figure Lengend Snippet: Representative images of YTHDF1, YTHDF2, and TIL expression in immunohistochemistry

Article Snippet: We used the following commercially available antibodies in recommended titrations: anti-YTHDF1 (1:100, 17479-1-AP, Proteintech, Rosemont, IL, USA ), YTHDF2 (1:400, ab170118, Abcam, Cambridge, UK), PD-1 (1:50, EH33, Cell Signaling Technology [CST], Danvers, MA, USA), CD8 (1:200, C8/144B, Nichirei, Tokyo, Japan), Foxp3 (1:100, 22510, Abcam), CD45RO (1:600, UCHL1, CST), PD-L1 (1:100, E1L3N; CST), epidermal growth factor receptor (EGFR) E746-A750 deletion specific (1:100, D6B6, CST), and EGFR L858R mutant specific (1:100, 43B2, CST).

Techniques: Expressing, Immunohistochemistry

Journal: Oncoimmunology

Article Title: YTHDF1 and YTHDF2 are associated with better patient survival and an inflamed tumor-immune microenvironment in non–small-cell lung cancer

doi: 10.1080/2162402X.2021.1962656

Figure Lengend Snippet: Level of YTHDF1 and YTHDF2 expression is elevated in NSCLC

Article Snippet: We used the following commercially available antibodies in recommended titrations: anti-YTHDF1 (1:100, 17479-1-AP, Proteintech, Rosemont, IL, USA ), YTHDF2 (1:400, ab170118, Abcam, Cambridge, UK), PD-1 (1:50, EH33, Cell Signaling Technology [CST], Danvers, MA, USA), CD8 (1:200, C8/144B, Nichirei, Tokyo, Japan), Foxp3 (1:100, 22510, Abcam), CD45RO (1:600, UCHL1, CST), PD-L1 (1:100, E1L3N; CST), epidermal growth factor receptor (EGFR) E746-A750 deletion specific (1:100, D6B6, CST), and EGFR L858R mutant specific (1:100, 43B2, CST).

Techniques: Expressing

Journal: Oncoimmunology

Article Title: YTHDF1 and YTHDF2 are associated with better patient survival and an inflamed tumor-immune microenvironment in non–small-cell lung cancer

doi: 10.1080/2162402X.2021.1962656

Figure Lengend Snippet: Comparison of clinical characteristics based on YTHDF1 and YTHDF2 expression in the tumor

Article Snippet: We used the following commercially available antibodies in recommended titrations: anti-YTHDF1 (1:100, 17479-1-AP, Proteintech, Rosemont, IL, USA ), YTHDF2 (1:400, ab170118, Abcam, Cambridge, UK), PD-1 (1:50, EH33, Cell Signaling Technology [CST], Danvers, MA, USA), CD8 (1:200, C8/144B, Nichirei, Tokyo, Japan), Foxp3 (1:100, 22510, Abcam), CD45RO (1:600, UCHL1, CST), PD-L1 (1:100, E1L3N; CST), epidermal growth factor receptor (EGFR) E746-A750 deletion specific (1:100, D6B6, CST), and EGFR L858R mutant specific (1:100, 43B2, CST).

Techniques: Expressing, Mutagenesis

Journal: Oncoimmunology

Article Title: YTHDF1 and YTHDF2 are associated with better patient survival and an inflamed tumor-immune microenvironment in non–small-cell lung cancer

doi: 10.1080/2162402X.2021.1962656

Figure Lengend Snippet: Survival curves according to YTHDF1 and YTHDF2 expression in patients with non–small-cell lung cancer (NSCLC)

Article Snippet: We used the following commercially available antibodies in recommended titrations: anti-YTHDF1 (1:100, 17479-1-AP, Proteintech, Rosemont, IL, USA ), YTHDF2 (1:400, ab170118, Abcam, Cambridge, UK), PD-1 (1:50, EH33, Cell Signaling Technology [CST], Danvers, MA, USA), CD8 (1:200, C8/144B, Nichirei, Tokyo, Japan), Foxp3 (1:100, 22510, Abcam), CD45RO (1:600, UCHL1, CST), PD-L1 (1:100, E1L3N; CST), epidermal growth factor receptor (EGFR) E746-A750 deletion specific (1:100, D6B6, CST), and EGFR L858R mutant specific (1:100, 43B2, CST).

Techniques: Expressing

Journal: Oncoimmunology

Article Title: YTHDF1 and YTHDF2 are associated with better patient survival and an inflamed tumor-immune microenvironment in non–small-cell lung cancer

doi: 10.1080/2162402X.2021.1962656

Figure Lengend Snippet: Univariate and multivariate Cox hazards model analyses for overall survival of patients with non–small-cell lung cancer

Article Snippet: We used the following commercially available antibodies in recommended titrations: anti-YTHDF1 (1:100, 17479-1-AP, Proteintech, Rosemont, IL, USA ), YTHDF2 (1:400, ab170118, Abcam, Cambridge, UK), PD-1 (1:50, EH33, Cell Signaling Technology [CST], Danvers, MA, USA), CD8 (1:200, C8/144B, Nichirei, Tokyo, Japan), Foxp3 (1:100, 22510, Abcam), CD45RO (1:600, UCHL1, CST), PD-L1 (1:100, E1L3N; CST), epidermal growth factor receptor (EGFR) E746-A750 deletion specific (1:100, D6B6, CST), and EGFR L858R mutant specific (1:100, 43B2, CST).

Techniques: Mutagenesis

Journal: Oncoimmunology

Article Title: YTHDF1 and YTHDF2 are associated with better patient survival and an inflamed tumor-immune microenvironment in non–small-cell lung cancer

doi: 10.1080/2162402X.2021.1962656

Figure Lengend Snippet: Univariate and multivariate Cox hazards model analyses for recurrence-free survival of patients with non–small-cell lung cancer

Article Snippet: We used the following commercially available antibodies in recommended titrations: anti-YTHDF1 (1:100, 17479-1-AP, Proteintech, Rosemont, IL, USA ), YTHDF2 (1:400, ab170118, Abcam, Cambridge, UK), PD-1 (1:50, EH33, Cell Signaling Technology [CST], Danvers, MA, USA), CD8 (1:200, C8/144B, Nichirei, Tokyo, Japan), Foxp3 (1:100, 22510, Abcam), CD45RO (1:600, UCHL1, CST), PD-L1 (1:100, E1L3N; CST), epidermal growth factor receptor (EGFR) E746-A750 deletion specific (1:100, D6B6, CST), and EGFR L858R mutant specific (1:100, 43B2, CST).

Techniques: Mutagenesis

Journal: Oncoimmunology

Article Title: YTHDF1 and YTHDF2 are associated with better patient survival and an inflamed tumor-immune microenvironment in non–small-cell lung cancer

doi: 10.1080/2162402X.2021.1962656

Figure Lengend Snippet: Expression of YTHDF1 and YTHDF2 is associated with tumor-infiltrating lymphocytes (TILs) in the stroma in patients with non–small-cell lung cancer

Article Snippet: We used the following commercially available antibodies in recommended titrations: anti-YTHDF1 (1:100, 17479-1-AP, Proteintech, Rosemont, IL, USA ), YTHDF2 (1:400, ab170118, Abcam, Cambridge, UK), PD-1 (1:50, EH33, Cell Signaling Technology [CST], Danvers, MA, USA), CD8 (1:200, C8/144B, Nichirei, Tokyo, Japan), Foxp3 (1:100, 22510, Abcam), CD45RO (1:600, UCHL1, CST), PD-L1 (1:100, E1L3N; CST), epidermal growth factor receptor (EGFR) E746-A750 deletion specific (1:100, D6B6, CST), and EGFR L858R mutant specific (1:100, 43B2, CST).

Techniques: Expressing

Journal: Oncoimmunology

Article Title: YTHDF1 and YTHDF2 are associated with better patient survival and an inflamed tumor-immune microenvironment in non–small-cell lung cancer

doi: 10.1080/2162402X.2021.1962656

Figure Lengend Snippet: YTHDF1 and YTHDF2 knockdown suppresses cell proliferation and partially induces apoptosis in lung cancer cells

Article Snippet: We used the following commercially available antibodies in recommended titrations: anti-YTHDF1 (1:100, 17479-1-AP, Proteintech, Rosemont, IL, USA ), YTHDF2 (1:400, ab170118, Abcam, Cambridge, UK), PD-1 (1:50, EH33, Cell Signaling Technology [CST], Danvers, MA, USA), CD8 (1:200, C8/144B, Nichirei, Tokyo, Japan), Foxp3 (1:100, 22510, Abcam), CD45RO (1:600, UCHL1, CST), PD-L1 (1:100, E1L3N; CST), epidermal growth factor receptor (EGFR) E746-A750 deletion specific (1:100, D6B6, CST), and EGFR L858R mutant specific (1:100, 43B2, CST).

Techniques:

Journal: Oncoimmunology

Article Title: YTHDF1 and YTHDF2 are associated with better patient survival and an inflamed tumor-immune microenvironment in non–small-cell lung cancer

doi: 10.1080/2162402X.2021.1962656

Figure Lengend Snippet: YTHDF1 and YTHDF2 knockdown increased tumoral PD-L1 expression in lung cancer cells

Article Snippet: We used the following commercially available antibodies in recommended titrations: anti-YTHDF1 (1:100, 17479-1-AP, Proteintech, Rosemont, IL, USA ), YTHDF2 (1:400, ab170118, Abcam, Cambridge, UK), PD-1 (1:50, EH33, Cell Signaling Technology [CST], Danvers, MA, USA), CD8 (1:200, C8/144B, Nichirei, Tokyo, Japan), Foxp3 (1:100, 22510, Abcam), CD45RO (1:600, UCHL1, CST), PD-L1 (1:100, E1L3N; CST), epidermal growth factor receptor (EGFR) E746-A750 deletion specific (1:100, D6B6, CST), and EGFR L858R mutant specific (1:100, 43B2, CST).

Techniques: Expressing

Journal: Clinical and Translational Medicine

Article Title: Downregulation of microRNA‐6125 promotes colorectal cancer growth through YTHDF2‐dependent recognition of N6‐methyladenosine‐modified GSK3β

doi: 10.1002/ctm2.602

Figure Lengend Snippet: MiR‐6125 inhibits the proliferation of CRC in vitro and in vivo. (A) MiR‐6125 and its control vector plasmid were transfected into SW480 and RKO cells to obtain stable transfected cells; transfection efficiency was detected by qPCR. (B and C) The effect of miR‐6125 overexpression on the anchorage‐independent growth of SW480 and RKO cells was analysed in a soft agar assay. (D and E) Effect of miR‐6125 on the proliferation rate of SW480 and RKO cells, as analysed by the ATP assay. (F and G) Effect of miR‐6125 on the proliferation rate of SW480 and RKO cells, as detected by the CCK8 assay. (H–K) EdU assay to examine the effect of miR‐6125 on the DNA replication activity of SW480 and RKO cells. (L–S) Nude mice were injected subcutaneously with SW480 (miR‐6125) or RKO (miR‐6125) and the corresponding control stable cell lines. After about 3 weeks, subcutaneous tumours were excised and photographed. A growth curve was drawn, the tumours were weighed, and miR‐6125 expression in the tumour was detected by qPCR. (T and U) Tumour samples were fixed and stained with haematoxylin and eosin. Differences in MKI67 expression were detected by immunohistochemical staining. An asterisk (*) indicates a significant difference at p < 0.05

Article Snippet: The tissue was incubated with the corresponding antibody at 4°C for at least 12 h. For IHC staining, antibodies specific for Cyclin D1 (Cell Signaling Technology, 2968), MKI67 (Abcam, ab16667), GSK3β (Cell Signaling Technology, 12456T), β‐catenin (Cell Signaling Technology, 8480P) and YTHDF2 (Proteintech, 24744‐1‐AP) were used.

Techniques: In Vitro, In Vivo, Plasmid Preparation, Transfection, Over Expression, Soft Agar Assay, ATP Assay, CCK-8 Assay, EdU Assay, Activity Assay, Injection, Stable Transfection, Expressing, Staining, Immunohistochemical staining