Journal: Frontiers in Cellular and Infection Microbiology

Article Title: CD147 mediates the adsorption of influenza A virus on the cell surface through direct interaction with HA

doi: 10.3389/fcimb.2025.1647283

Figure Lengend Snippet: CD147 was identified to positively regulate the proliferation of the H5N6 influenza virus. (A) Schematic diagram of the identification of host proteins that coprecipitated with influenza A viral protein HA. (I) Mass spectrometry was used to identify host proteins that coimmunoprecipitated with Ha-tagged influenza viral protein HA. (II) To identify host factors that affected viral replication, cells were transfected with plasmids to overexpress each of the candidate host genes and then infected with influenza virus. Cell viability, Virus titers and protein were then determined. (B) Viability of DF1 cells transfected with the indicated plasmids. (C) DF1 cells were transfected with the indicated plasmids, infected with H5N6 virus at an MOI of 0.001, the supernatant was collected for viral titer measurement, and the cells were harvested and analyzed via WB.

Article Snippet: The viability of DF1 and A549 cells following transfection, treatment with small molecule reagents, or knockout (KO) was assessed using a Cell-Counting Kit-8 (CCK-8) assay, according to the manufacturer’s instructions (MedChemExpress, #HY-K0301) ( ).

Techniques: Virus, Mass Spectrometry, Transfection, Infection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: CD147 mediates the adsorption of influenza A virus on the cell surface through direct interaction with HA

doi: 10.3389/fcimb.2025.1647283

Figure Lengend Snippet: Host membrane protein CD147 promotes the proliferation of influenza virus. (A, B) DF1 cells were transfected with chCD147-Flag or Flag-Empty Vector (EV), and infected with H5N6 virus 24 h after transfection. Supernatant was collected at different time points for TCID 50 measurement (A) , and the cells were harvested and analyzed via WB (B) . (C, D) DF1 cells were transfected with siRNA-chCD147 or siRNA-negative control (NC), and cells were harvested and analyzed via RT-qPCR (C) and Western blotting (WB) (D) . DF1 cells were infected with H5N6 virus 24 h after transfection with siRNAs. Supernatant was collected at different time points for TCID 50 measurement (E) , and the cells were harvested and analyzed via WB (F) . Cell viability was determined at the indicated time points (G) . (H, I) A549 cells were transfected with huCD147-Flag or Flag-EV, and infected with H5N6 virus 24 h after transfection. Supernatant was collected at different time points for TCID 50 measurement (H) , and the cells were harvested and analyzed via WB (I) . (J) The effect of knockdown in A549 cells was analyzed via WB. (K–M) CD147 knockdown A549 cells were infected with H1N1 (PR8) virus, and the supernatant was collected at different time points for TCID 50 measurement (K) . Cells were harvested and analyzed via WB (L) . Cell viability was determined at the indicated time points (M) . Error bars, mean ± SD of three experiments. All comparisons were tested by two-tailed Student’s t test; *, P, 0.05; **, P, 0.01; ***, P, 0.001; ****, P, 0.001.

Article Snippet: The viability of DF1 and A549 cells following transfection, treatment with small molecule reagents, or knockout (KO) was assessed using a Cell-Counting Kit-8 (CCK-8) assay, according to the manufacturer’s instructions (MedChemExpress, #HY-K0301) ( ).

Techniques: Membrane, Virus, Transfection, Plasmid Preparation, Infection, Negative Control, Quantitative RT-PCR, Western Blot, Knockdown, Two Tailed Test

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: CD147 mediates the adsorption of influenza A virus on the cell surface through direct interaction with HA

doi: 10.3389/fcimb.2025.1647283

Figure Lengend Snippet: CD147 interacted with HA. (A) HEK293T cells were transfected with plasmids encoding chCD147-Flag and H5-ACS-Ha. The lysates were subjected to anti-Flag IP and analyzed via Western blotting (WB). (B) DF1 cells were transfected with plasmids encoding H5-ACS-Ha. The lysates were subjected to anti Ha IP and analyzed via WB. (C) DF1 cells were transfected with plasmids encoding H5-ACS-Ha (H5N6 virus) and chCD147-Flag (avian source) and analyzed for colocalization of H5-ACS-Ha and chCD147-Flag. Scale bar, 20 mm. (D) HEK293T cells were transfected with plasmids encoding huCD147-Flag (human source) and H1-Ha (H1N1 virus), the lysates were subjected to anti-Flag IP and analyzed via WB. (E) HEK293T cells were transfected with plasmids encoding huCD147-Flag (human source) and H1-Ha (H1N1 virus), H5-ACS-Ha (H5N6 virus), H9-Ha (H9N2 virus) respectively, the lysates were subjected to anti-Flag IP and analyzed via WB. (F) Hela cells were transfected with H1-Ha and huCD147-Flag plasmid, and analyzed for colocalization of H1-Ha and huCD147-Flag. Scale bar, 20 mm. (G) A549 cells were infected with H1N1 (PR8), and analyzed for colocalization of CD147 and HA of H1N1 virus. Scale bar, 20 mm.

Article Snippet: The viability of DF1 and A549 cells following transfection, treatment with small molecule reagents, or knockout (KO) was assessed using a Cell-Counting Kit-8 (CCK-8) assay, according to the manufacturer’s instructions (MedChemExpress, #HY-K0301) ( ).

Techniques: Transfection, Western Blot, Virus, Plasmid Preparation, Infection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: CD147 mediates the adsorption of influenza A virus on the cell surface through direct interaction with HA

doi: 10.3389/fcimb.2025.1647283

Figure Lengend Snippet: CD147 promotes the absorption/endocytosis of influenza virus. (A, B) DF1 cells were transfected with chCD147-Flag and infected with H9N2 virus (MOI 50) 24 h after transfection at 4°C, after 1h of adsorption, they were transferred to 37°C for 15min and 60min. Samples of adsorbed and internalized cells were collected, vRNA was quantitatively analyzed by RT-qPCR (A) and Western blotting (WB) (B) . (C, D) DF1 cells were transfected with siRNA-chCD147 or siRNA-NC, and infected with H9N2 virus (MOI 50), washed with cold serum-free DMEM after 1 hour, the cells were cultured at 37°C for 1h, and then washed with acid PBS (pH 1.3), the vRNA was quantitatively analyzed by RT-qPCR (C) and Western blotting (WB) (D) . (E–H) A549 cells were transfected with huCD147-Flag or Flag-EV and CD147 knockdown A549 cells, infected with H1N1 (PR8) virus at 4°C, washed with cold serum-free Ham’s F-12 after 1 hour, the cells were cultured at 37°C for 1h, and then washed with acid PBS (pH 1.3), the vRNA was quantitatively analyzed by RT-qPCR (E, G) and Western blotting (WB) (F, H) . Error bars, mean ± SD of three experiments. All comparisons were tested by two-tailed Student’s t test; **, P, 0.01; ***, P, 0.001; ****, P, 0.001.

Article Snippet: The viability of DF1 and A549 cells following transfection, treatment with small molecule reagents, or knockout (KO) was assessed using a Cell-Counting Kit-8 (CCK-8) assay, according to the manufacturer’s instructions (MedChemExpress, #HY-K0301) ( ).

Techniques: Virus, Transfection, Infection, Adsorption, Quantitative RT-PCR, Western Blot, Cell Culture, Knockdown, Two Tailed Test

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: CD147 mediates the adsorption of influenza A virus on the cell surface through direct interaction with HA

doi: 10.3389/fcimb.2025.1647283

Figure Lengend Snippet: Virus infection, adsorption and glycosylation modified form of CD147. (A) DF1 cells were infected with H5N6 (JX) virus (MOI 0.001), and the cells were harvested and analyzed the change of protein level via WB. (C) A549 cells were infected with H1N1 (PR8) virus (MOI 0.01), and the cells were harvested and analyzed the change of protein level via WB. (E) A549 cells were exposed to PD1530356h, 6h 12h and 24h with different concentrations, and infected with PR8(MOI 10) at 4°C for 1h, the cells were harvested and analyzed the change of protein level via WB. (G) MDCK cells were exposed to PD1530356h 24 hours, and infected with H9N2 (MOI 10) at 4°C for 1h, the cells were harvested and analyzed the change of protein level via WB. (B, D, F, H) Gapdh was used as the control, Image J(NIH) was used to quantitatively analyze the gray level of protein bands, and CD147/Gapdh indicated the relative level of CD147. All comparisons were tested by two-tailed Student’s t test, the solid line represents the difference of HG-CD147 in different infection periods, and the dotted line represents the difference of LG-CD147 in different infection periods, blue * represents the difference of LG-CD147 in the same infection period, red * represents the difference of HG-CD147 in the same infection period, and black * represents the difference of total protein of CD147. *, P, 0.05; **, P, 0.01; ***, P, 0.001; ****, P, 0.001.

Article Snippet: The viability of DF1 and A549 cells following transfection, treatment with small molecule reagents, or knockout (KO) was assessed using a Cell-Counting Kit-8 (CCK-8) assay, according to the manufacturer’s instructions (MedChemExpress, #HY-K0301) ( ).

Techniques: Virus, Infection, Adsorption, Glycoproteomics, Modification, Control, Two Tailed Test

Journal: Heliyon

Article Title: Dexmedetomidine suppressed the biological behavior of RAW264.7 cells treated with LPS by down-regulating HOTAIR

doi: 10.1016/j.heliyon.2024.e27690

Figure Lengend Snippet: The primers of qRT-PCR used in this study.

Article Snippet: Antibodies were used, including rabbit anti-YTHDF1 monoclonal antibody (1:1000，Cell signaling technology), mouse anti-GAPDH monoclonal antibody (1:1000,Abcam).

Techniques:

Journal: Heliyon

Article Title: Dexmedetomidine suppressed the biological behavior of RAW264.7 cells treated with LPS by down-regulating HOTAIR

doi: 10.1016/j.heliyon.2024.e27690

Figure Lengend Snippet: Dexmedetomidine up-regulated the m6A level of HOTAIR in LPS-treated RAW264.7 cells. (A) The expression of HOTAIR m6A level was detect by MeRIP-qPCR. (B) HOTAIR was significantly enriched by YTHDF1 compared with IgG. (C) The expression of YTHDF1 was detect by qRT-PCR and WB in LPS-treated RAW264.7 cells. RAW264.7 cells were transfected with siYTHDF1 (300 nM) or pYTHDF1 (10 μg) in a 100-mm dish, and the cell lysate was divided into three parts: one for WB to determine the expression of YTHDF1 (D), and the other two for qRT-PCR to assess HOTAIR expression (E) and MeRIP-qPCR to determine the m6A level on HOTAIR (F).

Article Snippet: Antibodies were used, including rabbit anti-YTHDF1 monoclonal antibody (1:1000，Cell signaling technology), mouse anti-GAPDH monoclonal antibody (1:1000,Abcam).

Techniques: Expressing, Quantitative RT-PCR, Transfection

Journal: Discover. Oncology

Article Title: Systematic analysis of the expression profile and prognostic significance of m6A regulators and PD-L1 in hepatocellular carcinoma.

doi: 10.1007/s12672-022-00595-x

Figure Lengend Snippet: Fig. 3 Correlation between expression of m6A regulators and tumor staging (GEPIA) in patients with HCC. The mRNA expressions of YTHDF1/2, YTHDC1, METTL3 and RBM15 were significantly correlated with individual tumor stage

Article Snippet: The following primary antibodies were incubated for 12 h at 4 °C: GAPDH (1:10,000, ABclonal), YTHDF1 (1:1000, ABclonal), YTHDF2 (1:1000, ABclonal) and YTHDF3 (1:1000, ABclonal), KIAA1429(1:1000, Proteintech), METTL3(1:1000, Proteintech), RBM15(1:1000, Proteintech), ZC3H13(1:1000, Proteintech), METTL16(1:1000, Proteintech), ALKBH5(1:1000, Proteintech), METTL14(1:1000, Proteintech), WTAP(1:1000, Proteintech), HNRNPC(1:1000, Proteintech), YTHDC1(1:1000, Proteintech), YTHDC2(1:1000, Proteintech), FTO(1:1000, Proteintech).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Ebselen Suppresses Breast Cancer Tumorigenesis by Inhibiting YTHDF1-Mediated c-Fos Expression

doi: 10.3390/ijms26199416

Figure Lengend Snippet: YTHDF1 overexpression in breast cancer represents a viable therapeutic target for ebselen. ( a ) Comparison of RNA expression levels of the YTHDF gene family in breast cancer tissues and matched adjacent normal tissues, based on RNA-seq data from The Cancer Genome Atlas (TCGA). ( b ) Kaplan–Meier plot showing the association between YTHDF1 expression and progression-free survival in the TCGA breast cancer cohort. Patients with breast cancer ( n = 100 per group) were stratified into high and low YTHDF1 expression groups (data for panels ( a , b ) were retrieved from https://portal.gdc.cancer.gov/projects/TCGA-BRCA (accessed on 6 July 2025)). ( c ) Structural model showing ebselen binding to the YTH domain of YTHDF1, specifically near the m 6 A recognition pocket, forming a covalent bond with the Cys412 residue (PDB ID: 7PCU; structure retrieved from https://www.rcsb.org/ (accessed on 8 July 2025)). ( d ) Ebselen induces concentration-dependent cytotoxicity. In MCF7 cells. Cells were treated with the indicated concentrations of ebselen for 24 or 48 h, and cell viability was measured using the MTT assay.

Article Snippet: Antibodies against c-Fos (Clone 9F6; 2250; WB 1:1000), METTL3 (Clone E3F2A; 86132; WB 1:1000), Beclin-1 (3738; WB 1:1000), and YTHDF1 (86463S; WB 1:1000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Over Expression, Comparison, RNA Expression, RNA Sequencing, Expressing, Binding Assay, Residue, Concentration Assay, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Ebselen Suppresses Breast Cancer Tumorigenesis by Inhibiting YTHDF1-Mediated c-Fos Expression

doi: 10.3390/ijms26199416

Figure Lengend Snippet: Ebselen regulates YTHDF1-mediated c-Fos expression in MCF7 cells. ( a ) Volcano plot showing mRNAs upregulated by epidermal growth factor (EGF) treatment in a published RNA-seq dataset, with significantly upregulated transcripts highlighted in red. ( b , c ) Upregulation of FOS gene expression by EGF. MCF7 cells were cultured in serum-free medium for 24 h and treated with EGF for 1 h. Expression of FOS family genes was analyzed using real-time ( b ) or end-point ( c ) PCR with gene-specific primers. Data are presented as mean ± SD; n = 3. One-way analysis of variance (ANOVA), *** p < 0.001. ( d ) Ebselen treatment decreases FOS mRNA and protein levels in a concentration-dependent manner. MCF7 cells were treated with varying concentrations of ebselen in serum-free media for 24 h, followed by EGF stimulation. mRNA ( upper panel ) and protein ( lower panel ) expression was analyzed using end-point PCR and western blotting (WB) for the indicated target genes and proteins, respectively. ( e ) Knockout of YTHDF1 reduces FOS mRNA and c-Fos protein levels. MCF7 cells transfected with sgCtrl or sgYTHDF1 were serum-starved and treated with EGF. mRNA ( upper panel ) and protein ( lower panel ) expression was analyzed for the indicated target genes and proteins, respectively. ( f ) RNA immunoprecipitation (RIP) analysis using anti-YTHDF1 antibody confirmed the interaction between FOS mRNA and YTHDF1 protein. MCF7 cells were serum-starved and treated with varying concentrations of ebselen for 24 h, followed by EGF stimulation. Cell lysates were then subjected to RIP, and the associated RNA was analyzed via PCR. ( g , h ) Methyltransferase-like 3 (METTL3) regulates EGF-induced FOS expression. MCF7 cells transfected with sgCtrl or sgMETTL3 were serum-starved for 24 h ( g ). or wild-type cells were pre-treated with STM2457 (a METTL3 inhibitor) in serum-free medium ( h ). In both cases, cells were then stimulated with EGF for 1 h, followed by end-point PCR ( upper panel ) and WB ( lower panel ) analysis for the indicated target genes and proteins, respectively.

Article Snippet: Antibodies against c-Fos (Clone 9F6; 2250; WB 1:1000), METTL3 (Clone E3F2A; 86132; WB 1:1000), Beclin-1 (3738; WB 1:1000), and YTHDF1 (86463S; WB 1:1000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Expressing, RNA Sequencing, Gene Expression, Cell Culture, Concentration Assay, Western Blot, Knock-Out, Transfection, RNA Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Ebselen Suppresses Breast Cancer Tumorigenesis by Inhibiting YTHDF1-Mediated c-Fos Expression

doi: 10.3390/ijms26199416

Figure Lengend Snippet: Ebselen reduces FOS mRNA stability. ( a , b ) Effect of ebselen on FOS promoter activity. Schematic representation of the pGL5-FOS reporter construct containing the serum response element (indicated as SRE) consensus sequence in the promoter region of the luciferase reporter gene ( a ). MCF7 cells were transfected with the pGL5-FOS-luc reporter plasmid, followed by ebselen treatment and EGF stimulation ( b ). Firefly luciferase activity was then measured to assess FOS promoter activity. Data are presented as mean ± SD; n = 3. One-way ANOVA, ### p < 0.001. ( c , d ) Knockout of YTHDF1 reduces FOS mRNA stability. MCF7 cells transfected with sgControl or sgYTHDF1 were treated with actinomycin D (indicated as ActD) for the indicated time points. FOS mRNA expression was analyzed using end-point PCR ( c ). Band intensities were quantified via densitometry and normalized to that of GAPDH ( d ). Data are presented as mean ± SD; n = 3. Student’s t -test, * p < 0.05, *** p < 0.001 (sgControl versus sgYTHDF1 at corresponding time points), # p < 0.05 (sgControl, 0 min versus 60 min) ( d ). ( e , f ) Ebselen treatment reduces FOS mRNA stability. MCF7 cells were pretreated with ebselen (50 µM) for 24 h, followed by actinomycin treatment at the indicated time points. FOS mRNA expression was analyzed using end-point PCR ( e ), and band intensities were quantified via densitometry and normalized to that of GAPDH ( f ). Data are presented as mean ± SD; n = 3. Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001 (DMSO versus ebselen at corresponding time points), ## p < 0.01 (DMSO, 0 min versus 60 min).

Article Snippet: Antibodies against c-Fos (Clone 9F6; 2250; WB 1:1000), METTL3 (Clone E3F2A; 86132; WB 1:1000), Beclin-1 (3738; WB 1:1000), and YTHDF1 (86463S; WB 1:1000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Activity Assay, Construct, Sequencing, Luciferase, Transfection, Plasmid Preparation, Knock-Out, Expressing

Journal: Nature communications

Article Title: Adipocyte YTH N(6)-methyladenosine RNA-binding protein 1 protects against obesity by promoting white adipose tissue beiging in male mice.

doi: 10.1038/s41467-023-37100-z

Figure Lengend Snippet: Fig. 1 | Adipose YTHDF1 expression is reduced in obesity. a, b Immunoblot analysis of YTH family proteins in iWAT a and BAT b of mice fed with CD or HFD for 12 weeks. c, d mRNA levels of Yth family genes and thermogenesis-related genes in iWAT c and BAT d of CD- and HFD-fed mice. Data were presented as mean ± SEM

Article Snippet: Conditional Ythdf1 KO allele (Ythdf1CTL) was generated by Cyagen Biosciences (China).

Techniques: Expressing, Western Blot

Journal: Cancers

Article Title: m 6 A Methyltransferase KIAA1429 Regulates the Cisplatin Sensitivity of Gastric Cancer Cells via Stabilizing FOXM1 mRNA.

doi: 10.3390/cancers14205025

Figure Lengend Snippet: Figure 5. Effect of YTHDF1 on regulating FOXM1 expression. (A) RNA immunoprecipitation (RIP) indicated the direct binding within YTHDF1 and FOXM1 mRNA in AGS Cis-R or NCI-N87 Cis-R and their parental wild type cells. (B,C) The mRNA level of FOXM1 in YTHDF1 shRNA infected (B) or YTHDF1 overexpressed (C) AGS Cis-R or NCI-N87 Cis-R cells was assessed by QRT-PCR. (D) RNA immunoprecipitation (RIP) indicated the direct binding within YTHDF1 and FOXM1 mRNA in KIAA1429 shRNA infected AGS Cis-R or NCI-N87 Cis-R cells. (E,F) RNA decay rate assay demonstrated the FOXM1 mRNA half-lives upon the YTHDF1 knockdown in AGS Cis-R (E) or NCI- N87 Cis-R (F) cells. Data were detected at indicated timepoint with actinomycin D (Act D, 5 µg/mL) treatment. (G) Correlation analysis by Spearman’s rank correlation coefficient (GEPIA) showed the correlation between YTHDF1 and FOXM1 in the gastric cancer tissue specimens. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Plasmid for p65 overexpression was obtained from addgene [31]; pCMV6-Entry, KIAA1429, and YTHDF1 overexpression plasmids were purchased from Origene (Rockville, MD, USA).

Techniques: Expressing, RNA Immunoprecipitation, Binding Assay, shRNA, Infection, Quantitative RT-PCR, Knockdown