human fzd5 fc chimera  (R&D Systems)

Journal: Molecular Therapy. Nucleic Acids

Article Title: YTHDF1 Facilitates the Progression of Hepatocellular Carcinoma by Promoting FZD5 mRNA Translation in an m6A-Dependent Manner

doi: 10.1016/j.omtn.2020.09.036

Figure Lengend Snippet: Identifying FZD5 as a Target of YTHDF1 in HCC (A) Ribosome-seq of cells transfected with siYTHDF1 and control. (B) Overlapping ribosome-seq, PARCLIP-seq, and RIP-seq data. (C) KEGG analysis of putative targets of YTHDF1. (D) The expression abundance of FZD5, FZD7, FZD9, and WNT3 mRNAs in HCC tissues. (E) The transcription efficiency of most FZD5 were elevated in HCC tissues. (F) The fold change of expression levels and m6A levels of FZD5 after METTL14 knockdown in HepG2 cells. (G) IHC analysis of FZD5 in HCC tissues. (H) FZD5 protein expression was positively correlated with YTHDF1 protein expression in HCC tissues.

Article Snippet: Recombinant human FZD5 (rFZD5, 1617-FZC-050; R&D Systems, USA) was dissolved in PBS and added to cells at a final concentration of 10 μg/mL according to the manufacturer’s instructions.

Techniques: Transfection, Control, Expressing, Knockdown

Journal: Molecular Therapy. Nucleic Acids

Article Title: YTHDF1 Facilitates the Progression of Hepatocellular Carcinoma by Promoting FZD5 mRNA Translation in an m6A-Dependent Manner

doi: 10.1016/j.omtn.2020.09.036

Figure Lengend Snippet: YTHDF1 Promotes FZD5 Translation in an m6A-Dependent Manner (A) RIP-qRT-PCR analysis of the enrichment of FZD5 mRNA binding to YTHDF1 protein. (B) RIP-semiquantitative PCR analysis of the enrichment of FZD5 mRNA binding to YTHDF1 protein. (C) Western blot analysis of the effect of shYTHDF1 on FZD5 expression in HCC cells. (D) qRT-PCR analysis of the effect of shYTHDF1 on FZD5 expression in HCC cells. (E) Using CHX to test the stability of FZD5 protein in shYTHDF1 transfected HCC cells. (F) The location of potential m6A site in FZD5 mRNA. (G) MeRIP-qRT-PCR and semiquantitative PCR analysis of m6A site in FZD5 mRNA. (H) The luciferase assay in YTHDF1 overexpression HEK293T cells. (I) qRT-PCR analysis of the transfecting efficiency of si-METTL3. (J) Western blot analysis of the effect of si-METTL3#1 transfection on METTL3 and FZD5 expression. (K) The total m6A level of HCC cells after transfecting si-METTL3. (L) qRT-PCR analysis of the effect of transfecting si-METTL3#1 on FZD5 expression. (M) RIP-qRT-PCR and semiquantitative PCR analysis of the effect of si-METTL3 on the binding between FZD5 mRNA and YTHDF1 protein. ∗∗p < 0.01 and ∗∗∗p < 0.001.

Article Snippet: Recombinant human FZD5 (rFZD5, 1617-FZC-050; R&D Systems, USA) was dissolved in PBS and added to cells at a final concentration of 10 μg/mL according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Binding Assay, Western Blot, Expressing, Transfection, Luciferase, Over Expression

Journal: Molecular Therapy. Nucleic Acids

Article Title: YTHDF1 Facilitates the Progression of Hepatocellular Carcinoma by Promoting FZD5 mRNA Translation in an m6A-Dependent Manner

doi: 10.1016/j.omtn.2020.09.036

Figure Lengend Snippet: FZD5 Overexpression Effectively Reverses YTHDF1-Knockdown-Induced Inhibition of HCC Cells Progression (A) Western blot analysis of the transfection efficiency of rFZD5 in HCC cells. (B) Colony formation assay of HepG2 cells transfected with rFZD5 and shYTHDF1, respectively or corporately. (C) CCK-8 assay of HepG2 cells transfected with rFZD5 and shYTHDF1, respectively or corporately. (D) EdU incorporation assay of HepG2 cells transfected with rFZD5 and shYTHDF1, respectively or corporately. (E) Transwell migration assays of HepG2 cells transfected with rFZD5 and shYTHDF1, respectively or corporately. (F) Transwell Matrigel invasion assays of HepG2 cells transfected with rFZD5 and shYTHDF1, respectively or corporately. ns, not significant; ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001.

Article Snippet: Recombinant human FZD5 (rFZD5, 1617-FZC-050; R&D Systems, USA) was dissolved in PBS and added to cells at a final concentration of 10 μg/mL according to the manufacturer’s instructions.

Techniques: Over Expression, Knockdown, Inhibition, Western Blot, Transfection, Colony Assay, CCK-8 Assay, Migration

Journal: Molecular Therapy. Nucleic Acids

Article Title: YTHDF1 Facilitates the Progression of Hepatocellular Carcinoma by Promoting FZD5 mRNA Translation in an m6A-Dependent Manner

doi: 10.1016/j.omtn.2020.09.036

Figure Lengend Snippet: YTHDF1 Promotes HCC Progression through the WNT/β-Catenin Signaling Pathway (A) Western blot analysis of β-catenin and c-MYC in HCC cells transfected with rFZD5 and shYTHDF1, respectively or corporately. (B) The concentrations of extracellular calcium in shYTHDF1 transfected HCC cells. (C) The phosphorylated JNK levels in shYTHDF1 transfected HCC cells. (D) H&E-staining and IHC analysis of YTHDF1, FZD5, β-catenin, and c-MYC in xenograft tumor samples. (E) The positive correlations between FZD5, β-catenin, c-MYC, and YTHDF1 expression in TCGA database of HCC. (F) The illustration summarizes the role of YTHDF1 in the regulatory network in HCC cells.

Article Snippet: Recombinant human FZD5 (rFZD5, 1617-FZC-050; R&D Systems, USA) was dissolved in PBS and added to cells at a final concentration of 10 μg/mL according to the manufacturer’s instructions.

Techniques: Western Blot, Transfection, Staining, Expressing