Journal: bioRxiv

Article Title: YTHDF3 promotes the progression of gastric cancer by activating Wnt/β-catenin signaling pathway via targeting NEK7

doi: 10.1101/2025.01.25.634861

Figure Lengend Snippet: (A-C) Expression of YTHDF3 in the TCGA database, as well as the GSE54129 and GSE66229 datasets. (D) YTHDF3 is highly expressed in STAD tissues through the analysis of the online databases GEPIA2. (E) The expression levels of YTHDF3 mRNA in 12 pairs of GC and adjacent normal tissue samples were detected by qRT-PCR. (F) Representative images showing YTHDF3 protein expression levels in 50 pairs of GC and adjacent normal tissue samples, as detected by IHC. Scale bar, 20 μm. Data are presented as means ± SD. * P <0.05

Article Snippet: YTHDF3 antibody (diluted 1:50; Proteintech; Catalog number: 25537-1-AP) and NEK7 antibody (diluted 1:50; Santa Cruz Biotechnology; Catalog number: sc-393539) were then added to the cells and incubated for an entire night at 4°C.

Techniques: Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: YTHDF3 promotes the progression of gastric cancer by activating Wnt/β-catenin signaling pathway via targeting NEK7

doi: 10.1101/2025.01.25.634861

Figure Lengend Snippet: (A, B) The expression of YTHDF3 mRNA in HGC-27 and AGS cells was detected by qRT-PCR. (C, D) The expression of YTHDF3 protein in HGC-27 and AGS was detected by WB. (E, F) The effect of YTHDF3 knockdown on the proliferation of HGC-27 and AGS cells was determined by the CCK-8 assay. (G, H) The effect of YTHDF3 knockdown on the migration of HGC-27 and AGS cells was assessed by the wound healing assay. Scale bar, 200 μm. (K, L) The effect of YTHDF3 knockdown on the migration of HGC-27 and AGS cells was evaluated using the Transwell migration assay. Scale bar, 100 μm. (M, N) The effect of YTHDF3 knockdown on the invasion of HGC-27 and AGS cells was assessed using Transwell invasion assay. Scale bar, 100 μm. (I, J) The effect of YTHDF3 knockdown on the apoptosis of HGC-27 and AGS cells was determined by flow cytometry. Data are presented as means ± SD. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001

Article Snippet: YTHDF3 antibody (diluted 1:50; Proteintech; Catalog number: 25537-1-AP) and NEK7 antibody (diluted 1:50; Santa Cruz Biotechnology; Catalog number: sc-393539) were then added to the cells and incubated for an entire night at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, CCK-8 Assay, Migration, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay, Flow Cytometry

Journal: bioRxiv

Article Title: YTHDF3 promotes the progression of gastric cancer by activating Wnt/β-catenin signaling pathway via targeting NEK7

doi: 10.1101/2025.01.25.634861

Figure Lengend Snippet: (A, B) The expression of YTHDF3 mRNA in MKN7 and MKN74 was detected by qRT-PCR. (C, D) The expression of YTHDF3 protein in MKN7 and MKN74 was detected by WB. (E, F) The effect of YTHDF3 overexpression on the proliferation of MKN7 and MKN74 cells was assessed using the CCK-8 assay. (G, H) The effect of YTHDF3 overexpression on the migration of MKN7 and MKN74 cells was evaluated through a wound healing assay. Scale bar, 200 μm. (K, L) The effect of YTHDF3 overexpression on the migration of MKN7 and MKN74 cells was determined utilizing a Transwell migration assay. Scale bar, 100 μm. (M, N) The effect of YTHDF3 overexpression on the invasion of MKN7 and MKN74 cells was assessed via a Transwell invasion assay. Scale bar, 100 μm. (I, J) The effect of YTHDF3 overexpression on the apoptosis of MKN7 and MKN74 cells was analyzed by flow cytometry. Data are presented as means ± SD. ** P <0.01, *** P <0.001, **** P <0.0001

Article Snippet: YTHDF3 antibody (diluted 1:50; Proteintech; Catalog number: 25537-1-AP) and NEK7 antibody (diluted 1:50; Santa Cruz Biotechnology; Catalog number: sc-393539) were then added to the cells and incubated for an entire night at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Over Expression, CCK-8 Assay, Migration, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay, Flow Cytometry

Journal: bioRxiv

Article Title: YTHDF3 promotes the progression of gastric cancer by activating Wnt/β-catenin signaling pathway via targeting NEK7

doi: 10.1101/2025.01.25.634861

Figure Lengend Snippet: (A) Differential expression genes (DEGs) heat map identified by RNA-seq. (B) The number of DEGs identified by RNA-seq. (C) Overlap analysis of RNA-seq, YTHDF3-cor and STAD-DEGs-up identified genes. (D) Expression of YTHDF3 in GC paired tissue cohort from the TCGA database. (E) Subcellular localization of NEK7 from GeneCards. (F) The survival analysis of NEK7 expression levels and overall survival in patients with GC. (G, H) the interaction between YTHDF3 and NEK7 was verified by Co-IF (G) and Co-IP (H) experiments. Scale bar, 10μm. (I) qRT-PCR was used to detect NEK7 mRNA expression after YTHDF3 knockdown in HGC-27 cells. (J) WB was used to detect NEK7 protein expression after YTHDF3 knockdown in HGC-27 cells. (K) qRT-PCR was used to detect NEK74 mRNA expression after YTHDF3 overexpression in MKN74 cells. (L) WB was used to detect NEK7 protein expression after YTHDF3 overexpression in MKN74 cells. Data are presented as means ± SD. *** P <0.001, **** P <0.0001

Article Snippet: YTHDF3 antibody (diluted 1:50; Proteintech; Catalog number: 25537-1-AP) and NEK7 antibody (diluted 1:50; Santa Cruz Biotechnology; Catalog number: sc-393539) were then added to the cells and incubated for an entire night at 4°C.

Techniques: Quantitative Proteomics, RNA Sequencing, Expressing, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Knockdown, Over Expression

Journal: bioRxiv

Article Title: YTHDF3 promotes the progression of gastric cancer by activating Wnt/β-catenin signaling pathway via targeting NEK7

doi: 10.1101/2025.01.25.634861

Figure Lengend Snippet: (A) The expression ofNEK7 mRNA in GC cell lines and GES-1 was detected by qRT-PCR. (B) The expression of NEK7 mRNA in 18 pairs of GC paired tissues was detected by qRT-PCR. (C) The basal expression of NEK7 protein in cell lines was detected by WB. (D) The expression of YTHDF3 protein in 44 pairs of GC paired tissues was detected by IHC. Scale bar, 50 μm. (E-F) Four siRNA-NEK7 sequences were transfected simultaneously into MKN74 cells. (G-I) The relative expression level of YTHDF3 mRNA and protein were detected by qRT-PCR and WB after instantaneous co-transfection of YTHDF3 siRNA and NEK7 overexpression plasmid in HGC-27. (I-J) The relative expression level of YTHDF3 mRNA and protein were detected by qRT-PCR and WB after instantaneous co-transfection of NEK7 siRNA and YTHDF3 overexpression plasmid in MKN74. Data are presented as means ± SD. *** P <0.001, **** P <0.0001

Article Snippet: YTHDF3 antibody (diluted 1:50; Proteintech; Catalog number: 25537-1-AP) and NEK7 antibody (diluted 1:50; Santa Cruz Biotechnology; Catalog number: sc-393539) were then added to the cells and incubated for an entire night at 4°C.

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

Journal: bioRxiv

Article Title: YTHDF3 promotes the progression of gastric cancer by activating Wnt/β-catenin signaling pathway via targeting NEK7

doi: 10.1101/2025.01.25.634861

Figure Lengend Snippet: (7A) KEGG enrichment result of the differentially expressed gene sets from the RNA-seq. (7B) The expression levels of Wnt/β-catenin signaling pathway related proteins were detected by WB after instantaneous co-transfection of YTHDF3 siRNA and NEK7 overexpression plasmid in HGC-27. (C, E, G, I, K) The effects of NEK7 on YTHDF3 in HGC-27 cells were detected by CCK-8 assay (C), colony formation assay (E), wound healing assay (G),Scale bar, 200 μm, Transwell migration assay (I) and Transwell invasion assay (K). Scale bar, 100 μm. (D, F, H, J, L) The effects of NEK7 on YTHDF3 in MKN74 cells were detected by CCK-8 assay (D), colony formation assay (F), wound healing assay H), Scale bar, 200 μm, Transwell migration assay (J) and Transwell invasion assay (L). Scale bar, 100 μm.. Data are presented as means ± SD. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001

Article Snippet: YTHDF3 antibody (diluted 1:50; Proteintech; Catalog number: 25537-1-AP) and NEK7 antibody (diluted 1:50; Santa Cruz Biotechnology; Catalog number: sc-393539) were then added to the cells and incubated for an entire night at 4°C.

Techniques: RNA Sequencing, Expressing, Cotransfection, Over Expression, Plasmid Preparation, CCK-8 Assay, Colony Assay, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay

Journal: Cancer cell international

Article Title: Stabilization of UCA1 by N6-methyladenosine RNA methylation modification promotes colorectal cancer progression.

doi: 10.1186/s12935-021-02288-x

Figure Lengend Snippet: Fig. 6 IGF2BP2 serves as a reader for m6A modified UCA1. A The workflow of in vivo S1m-tagging RNA pulldown assay. B Detection of the YTHDF1, YTHDF2 and YTHDF3 by western blot, after in vivo RNA pulldown. C Detection of the IGF2BP1, IGF2BP2 and IGF2BP3 by western blot, after in vivo RNA pulldown. D The interaction between UCA1 and IGF2BP2 was confirmed by RIP assay. Error bars represent SD, n = 3, ***P < 0.005. E Mutation on m6A motif of UCA1 impairs the interaction of IGF2BP2 with UCA1. Left, the schematic of the A to C mutation on UCA1 m6A motif; right, the representative western blot showing that the mutant UCA1revealed little interaction with IGF2BP2 and UCA1, in contrast to WT UCA1

Article Snippet: WTAP (56501) and METTL3 (96391) were purchased from Cell Signaling Technology (Danvers, MA), YTHDF1 (17479-1-AP), YTHDF2 (24744-1-AP), YTHDF3 (25537- 1-AP), IGF2BP1 (22803-1-A), IGF2BP2 (11601-1-AP), IGF2BP3 (14642-1-AP) were from Proteintech (Chicago, IL, USA).

Techniques: Modification, In Vivo, Western Blot, Mutagenesis

Journal: MedComm

Article Title: N6‐methyladenosine reader YTHDF3‐mediated zinc finger protein 41 inhibits hepatocellular carcinoma progression by transcriptional repression of Snail

doi: 10.1002/mco2.763

Figure Lengend Snippet: YTHDF3‐mediated m 6 A modification of ZFP41 mRNA and decays its mRNA stability. (A) qPCR results showed that efficiencies of siRNA‐mediated knockdown of common m 6 A regulators in MHCC‐97H cells. (B) qPCR results showed that expression of ZFP41 after silencing these m 6 A regulators in MHCC‐97H cells. (C) Overexpression of YTHDF3 notably suppress ZFP41 expression on transcription level in MHCC‐97H and Hep3B cells. (D) Knockdown of YTHDF3 obviously increase ZFP41 expression on mRNA level in MHCC‐97H and Hep3B cells. (E) Western blots results demonstrated that the ZFP41 protein level in MHCC‐97H and HLF cells after silencing YTHDF3 with siRNA of YTHDF3. (F) Western blots results demonstrated that the ZFP41 protein level in Hep3B cells after overexpression of YTHDF3. (G) The diagram of the potential site of m 6 A modification on the CDS (Coding Sequence) area. (H) The luciferase activity in both MHCC‐97H and Hep3B cells cotransfected with relative plasmids. (I) MeRIP results showed that ZFP41‐wt or ZFP41‐mut in both MHCC‐97H and Hep3B cells. (J) The rate of ZFP41 mRNA degradation in MHCC‐97H and Hep3B cells with YTHDF3 overexpression or knockdown. (K) The binding of ZFP41 mRNA and YTHDF3 was tested in MHCC‐97H and Hep3B cells by RIP‐qPCR analyses. (L) The relationship between ZFP41 and YTHDF3 was confirmed by RNA pull‐down assay.

Article Snippet: Antibodies for YTHDF3 (25537‐1‐AP), E‐cadherin (20874‐1‐AP), N‐cadherin (22018‐1‐AP), and GAPDH (60004‐1‐Ig) were supplied by Proteintech.

Techniques: Modification, Knockdown, Expressing, Over Expression, Western Blot, Sequencing, Luciferase, Activity Assay, Binding Assay, Pull Down Assay

Journal: MedComm

Article Title: N6‐methyladenosine reader YTHDF3‐mediated zinc finger protein 41 inhibits hepatocellular carcinoma progression by transcriptional repression of Snail

doi: 10.1002/mco2.763

Figure Lengend Snippet: Schematic diagram of the mechanism showing the roles played by ZFP41 in HCC cells. In normal liver cells, YTHDF3 shows low expression, leading to a reduction in the m6A modification of ZFP41. This results in increased synthesis of ZFP41 mRNA and an enhanced transcriptional inhibitory effect on Snail. In contrast, in HCC cells, the expression of YTHDF3 is significantly elevated, which enhances the m6A modification of ZFP41. This promotes degradation of ZFP41 mRNA, consequently weakening the transcriptional inhibitory effect on Snail. Ultimately, this facilitates activation of the EMT pathway and promotes proliferation and metastasis of HCC.

Article Snippet: Antibodies for YTHDF3 (25537‐1‐AP), E‐cadherin (20874‐1‐AP), N‐cadherin (22018‐1‐AP), and GAPDH (60004‐1‐Ig) were supplied by Proteintech.

Techniques: Expressing, Modification, Activation Assay