Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulated YTHDC1 mediates trophoblastic dysfunction inducing preterm birth in ART conceptions through enhanced RPL37 translation

doi: 10.1007/s00018-024-05467-x

Figure Lengend Snippet: YTHDC1 is upregulated in human ART-preterm placentas. ( A ) qRT-PCR detection of the expression level of YTHDC1 in placentas conceived by assisted reproductive technology (ART). Preterm: preterm placentas ( n = 12); Term: full-term placentas ( n = 17). ( B ) Western blotting detection of YTHDC1 in the placentas conceived by ART. Preterm ( n = 6), Term ( n = 6). ( C , D ) Immunohistochemistry (IHC) images and quantification of YTHDC1 positive staining areas in placentas conceived by ART. Preterm ( n = 6), Term ( n = 6). Scale bar, 200 μm and 50 μm. Data are shown as means ± SD. ** P < 0.01

Article Snippet: After twice washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min, and then blocked by Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min. After that, cells were incubated with YTHDC1 primary antibody (1:200, NBP1-81353, NOVUS) overnight at 4 °C prior to incubation with fluorescence-labelled secondary antibody (1:200, R37120, Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Beyotime), and images were visualized using confocal microscopy.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemistry, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulated YTHDC1 mediates trophoblastic dysfunction inducing preterm birth in ART conceptions through enhanced RPL37 translation

doi: 10.1007/s00018-024-05467-x

Figure Lengend Snippet: YTHDC1 knockdown inhibits the proliferation, migration, and invasion of trophoblastic cells. ( A ) Knockdown efficiency of YTHDC1 siRNA in HTR-8/SVneo and JAR cells as assessed by qRT-PCR. ( B ) Knockdown efficiency of YTHDC1 siRNA in HTR-8/SVneo and JAR cells as assessed by Western blotting. ( C ) Knockdown effects of YTHDC1 on the cellular viability of HTR-8/SVneo and JAR cells as detected by CCK-8. ( D ) Knockdown effects of YTHDC1 on the proliferation of HTR-8/SVneo and JAR cells as detected by colony formation assays. ( E , F ) Knockdown effects of YTHDC1 on the proliferation of HTR-8/SVneo and JAR cells as detected by EdU assay. Scale bar, 100 μm. ( G , H ) Cell apoptotic rate as analyzed by flow cytometry. ( I , J ) Knockdown effects of YTHDC1 on the migration (I) and invasion (J) of HTR-8/SVneo and JAR cells as detected by Transwell assay. Scale bar, 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After twice washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min, and then blocked by Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min. After that, cells were incubated with YTHDC1 primary antibody (1:200, NBP1-81353, NOVUS) overnight at 4 °C prior to incubation with fluorescence-labelled secondary antibody (1:200, R37120, Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Beyotime), and images were visualized using confocal microscopy.

Techniques: Knockdown, Migration, Quantitative RT-PCR, Western Blot, CCK-8 Assay, EdU Assay, Flow Cytometry, Transwell Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulated YTHDC1 mediates trophoblastic dysfunction inducing preterm birth in ART conceptions through enhanced RPL37 translation

doi: 10.1007/s00018-024-05467-x

Figure Lengend Snippet: Identification of the signaling pathways regulated by YTHDC1 in trophoblastic cells. ( A ) Heatmap of differentially expressed genes (DEGs) identified by RNA-seq in HTR-8/SVneo cells. ( B ) Volcano plot of DEGs. ( C ) GO enrichment analysis of DEGs. ( D ) GSEA analysis of DEGs. ( E , F ) The knockdown (E) and overexpression (F) effects of YTHDC1 on the expression of DKK1, PIK3R3, and LIFR in HTR-8/SVneo and JAR cells as detected by Western blotting. ( G ) CCK-8 assay of JAR cells transfected with a YTHDC1 overexpression plasmid and PIK3R3 siRNA. ( H , I ) Colony formation assay of JAR cells transfected with a YTHDC1 overexpression plasmid and PIK3R3 siRNA. ( J , K ) Transwell assay about migration of JAR cells transfected with a YTHDC1 overexpression plasmid and PIK3R3 siRNA. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: After twice washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min, and then blocked by Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min. After that, cells were incubated with YTHDC1 primary antibody (1:200, NBP1-81353, NOVUS) overnight at 4 °C prior to incubation with fluorescence-labelled secondary antibody (1:200, R37120, Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Beyotime), and images were visualized using confocal microscopy.

Techniques: RNA Sequencing Assay, Knockdown, Over Expression, Expressing, Western Blot, CCK-8 Assay, Transfection, Plasmid Preparation, Colony Assay, Transwell Assay, Migration

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulated YTHDC1 mediates trophoblastic dysfunction inducing preterm birth in ART conceptions through enhanced RPL37 translation

doi: 10.1007/s00018-024-05467-x

Figure Lengend Snippet: YTHDC1 accelerates protein synthesis in trophoblastic cells. ( A ) The m 6 A motif detected by MEME algorithm analysis in identified mRNAs by MeRIP-seq in HTR-8/SVneo cells. ( B ) Metagene profiles of m 6 A enriched regions across mRNA segments in HTR-8/SVneo cells. ( C ) The distribution of m 6 A modified sites within mRNAs in HTR-8/SVneo cells. ( D ) GO enrichment analysis of the DEGs identified by RIP-seq. ( E ) Overlapping analysis of genes identified by RNA-seq, RIP-seq, and MeRIP-seq in HTR-8/SVneo and JAR cells. RNA-seq analysis was conducted on YTHDC1 knockdown HTR-8/SVneo cells. ( F ) Functional annotation of the overlapping genes. ( G ) IF staining of YTHDC1 in HTR-8/SVneo and JAR cells. Scale bar, 25 μm. ( H - J ) The effects of YTHDC1 knockout ( H , I ) or overexpression (J) on protein synthesis in HTR-8/SVneo and JAR cells detected by OP-Puro assays. Scale bar, 50 μm. ( K , L ) The effects of YTHDC1 knockdown ( K ) or overexpression (L) on de novo protein synthesis in HTR-8/SVneo and JAR cells as detected by SUnSET assays. ( M ) Polysome profiling of YTHDC1 overexpressed JAR cells

Article Snippet: After twice washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min, and then blocked by Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min. After that, cells were incubated with YTHDC1 primary antibody (1:200, NBP1-81353, NOVUS) overnight at 4 °C prior to incubation with fluorescence-labelled secondary antibody (1:200, R37120, Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Beyotime), and images were visualized using confocal microscopy.

Techniques: Modification, RNA Sequencing Assay, Knockdown, Functional Assay, Staining, Knock-Out, Over Expression

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulated YTHDC1 mediates trophoblastic dysfunction inducing preterm birth in ART conceptions through enhanced RPL37 translation

doi: 10.1007/s00018-024-05467-x

Figure Lengend Snippet: YTHDC1 regulates RPL37 translation in an m 6 A-dependent manner. ( A ) Relative mRNA level of RPL37 and eIF4G in human ART-preterm and ART-term placentas as detected by qRT-PCR. Preterm: preterm placentas ( n = 12); Term: full-term placentas ( n = 17). ( B , C ) Relative mRNA level of RPL37 and eIF4G in HTR-8/SVneo and JAR cells upon YTHDC1 knockdown ( B ) or overexpression ( C ) as detected by qRT-PCR. ( D , E ) Relative protein levels of RPL37 and eIF4G in HTR-8/SVneo and JAR cells upon YTHDC1 knockdown ( D ) or overexpression ( E ) as detected by Western blotting. ( F ) Integrative genomics viewer (IGV) tracks of m 6 A peaks and YTHDC1 binding peaks across RPL37 transcript. ( G ) qRT-PCR analysis of RPL37 in the MeRIP products. MeRIP carried out in HTR-8/SVneo and JAR cells with antibody against m 6 A, or the control unimmunized IgG. ( H ) qRT-PCR analysis of RPL37 in the RIP products. RIP carried out in HTR-8/SVneo and JAR cells with antibody against YTHDC1, or the control unimmunized IgG. ( I , J ) Schematic representation of wild-type (RPL37-WT) and mutant (RPL37-MUT) RPL37 luciferase reporters. ( K ) Luciferase activities of RPL37-WT or RPL37-MUT measured in 293T cells with or without YTHDC1 knockout. ( L ) Western blotting detection of YTHDC1 and RPL37. HTR-8/SVneo and JAR cells were co-transfected with YTHDC1 siRNA and RPL37 overexpression plasmid. ( M , N ) Rescue assay of cellular viability as detected with CCK-8. HTR-8/SVneo (M) and JAR cells ( N ) were co-transfected with YTHDC1 siRNA and RPL37 overexpression plasmid. ( O , P ) Rescue assay of colony formation. ( Q ) Rescue assay of SUnSET experiment. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After twice washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min, and then blocked by Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min. After that, cells were incubated with YTHDC1 primary antibody (1:200, NBP1-81353, NOVUS) overnight at 4 °C prior to incubation with fluorescence-labelled secondary antibody (1:200, R37120, Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Beyotime), and images were visualized using confocal microscopy.

Techniques: Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Binding Assay, Control, Mutagenesis, Luciferase, Knock-Out, Transfection, Plasmid Preparation, Rescue Assay, CCK-8 Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulated YTHDC1 mediates trophoblastic dysfunction inducing preterm birth in ART conceptions through enhanced RPL37 translation

doi: 10.1007/s00018-024-05467-x

Figure Lengend Snippet: Estradiol (E2) promotes YTHDC1 transcription through RXRA. ( A ) Luciferase assay of the YTHDC1 promoter. HEK-293T cells were transfected with reporter plasmids containing a series of YTHDC1 promoter truncations. ( B ) qRT-PCR quantification of potential transcription factors, RXRA and GTF2I , in human placental tissues. The potential transcription factors about the human YTHDC1 promoter between − 1000 to -900 bp are predicted by the ALGGEN-PROMO version 8.3 online tool of TRANSFAC. ( C ) Relative mRNA level of YTHDC1 as detected by qRT-PCR in HTR-8/SVneo and JAR cells upon knockdown of RXRA or GTF2I. ( D ) Western blotting analysis of YTHDC1 in HTR-8/SVneo and JAR cells upon knockdown of RXRA or GTF2I . ( E ) Luciferase assay about the effect of RXRA on the transcription of YTHDC1 . HEK-293T cells were transfected with RXRA siRNA or overexpression plasmid. ( F ) Alignment of ChIP-seq data from HTR-8/SVneo to the hg38 genome. The box showed the enrichment of RXRA and H3K27ac in the promoter of YTHDC1 . ( G ) ChIP-qPCR assay about the binding of RXRA to the promoter of YTHDC1 in HTR-8/SVneo and JAR cells. ( H ) Western blotting analysis of RXRA, YTHDC1, RPL37. HTR-8/SVneo and JAR cells were treated with E2 at the indicated concentrations for 24 h. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After twice washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min, and then blocked by Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min. After that, cells were incubated with YTHDC1 primary antibody (1:200, NBP1-81353, NOVUS) overnight at 4 °C prior to incubation with fluorescence-labelled secondary antibody (1:200, R37120, Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Beyotime), and images were visualized using confocal microscopy.

Techniques: Luciferase, Transfection, Quantitative RT-PCR, Knockdown, Western Blot, Over Expression, Plasmid Preparation, ChIP-sequencing, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulated YTHDC1 mediates trophoblastic dysfunction inducing preterm birth in ART conceptions through enhanced RPL37 translation

doi: 10.1007/s00018-024-05467-x

Figure Lengend Snippet: High E2 exposure elevates YTHDC1 expression and knockdown of YTHDC1 postpones murine preterm delivery in vivo. ( A - D ) Murine model of high E2 exposure. C57BL/6J mice were pretreated with E2 or corn oil, and preterm delivery was induced at E15.5d by RU486, while controls were treated with PBS (Oil-MF, n = 6; Oil-PBS, n = 6, E2-MF, n = 5; E2-PBS, n = 6). ( A ) The schematic diagram. ( B ) Relative mRNA level of YTHDC1 in the placentas as detected by qRT-PCR. ( C ) Western blotting detection of the expression of YTHDC1, RPL37, and PIK3R3 in the placentas. ( D ) Representative IHC staining images of YTHDC1, RPL37 and PIK3R3 in the placentas. Scale bar, 200 μm and 50 μm. ( E-H ) The schematic diagram for murine model of YTHDC1 siRNA treatment. Pregnant C57BL/6J mice were was intravenously administered with siYTHDC1 or siNC via the tail vein, and preterm delivery was induced at E15.5d by RU486. ( E ) The schematic diagram. ( F ) The initiation time of preterm labor (siYTHDC1, n = 9; siNC, n = 7). ( G ) Western blotting detection of YTHDC1, RPL37, and PIK3R3 in the placentas. ( H ) Representative IHC staining images of YTHDC1, RPL37, Ki-67, 11-β-HSD2 and CGB in the placentas. Scale bar, 50 μm. ** P < 0.01

Article Snippet: After twice washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min, and then blocked by Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min. After that, cells were incubated with YTHDC1 primary antibody (1:200, NBP1-81353, NOVUS) overnight at 4 °C prior to incubation with fluorescence-labelled secondary antibody (1:200, R37120, Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Beyotime), and images were visualized using confocal microscopy.

Techniques: Expressing, Knockdown, In Vivo, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Upregulated YTHDC1 mediates trophoblastic dysfunction inducing preterm birth in ART conceptions through enhanced RPL37 translation

doi: 10.1007/s00018-024-05467-x

Figure Lengend Snippet: The schematic diagram about working model of YTHDC1. Estradiol (E2) promoted YTHDC1 expression through RXRA upregulation. Elevated YTHDC1 upregulated the expression of RPL37 by promoting the binding of YTHDC1 to m 6 A-modified RPL37 mRNA, thereby augmenting total mRNA translation and indirectly modulated the JAK/STAT/PIK3R3 signaling pathway in trophoblastic cells

Article Snippet: After twice washing with PBS, cells were permeabilized with 0.25% Triton X-100 for 15 min, and then blocked by Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 30 min. After that, cells were incubated with YTHDC1 primary antibody (1:200, NBP1-81353, NOVUS) overnight at 4 °C prior to incubation with fluorescence-labelled secondary antibody (1:200, R37120, Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Beyotime), and images were visualized using confocal microscopy.

Techniques: Expressing, Binding Assay, Modification

Journal: Experimental & Molecular Medicine

Article Title: Loss of YTHDC1 m 6 A reading function promotes invasiveness in urothelial carcinoma of the bladder

doi: 10.1038/s12276-024-01377-x

Figure Lengend Snippet: a Kaplan‒Meier ten-year overall survival analysis of patients with different levels of YTHDC1 using data from the Mannheim University Hospital cohort. Statistical significance was assessed by the log-rank test. b Comparison of YTHDC1 mRNA expression between NMIBC ( n = 18) and MIBC ( n = 80) patients in the Mannheim University Hospital cohort. ** p -value < 0.01, Mann‒Whitney U test. c YTHDC1 mRNA expression levels in NMIBC compared with MIBC samples from the published Fudan cohort and UROMOL cohorts. *** p -value < 0.001, ** p -value < 0.01, Mann‒Whitney U test. d Representative H&E staining (upper panel) and IHC (lower panel) results showing YTHDC1 expression across different stages of bladder cancer. Scale bar = 100 μm. Quantitative analyses of the YTHDC1 IHC assays are shown in the right panels, which were performed via the IRS method and compared between paratumoral and tumor tissues (above), as well as between NMIBC and MIBC tissues (below). *** p -value < 0.001, ** p -value < 0.01, Mann‒Whitney U test. e Pearson correlation analyses between YTHDC1 expression (Log 2 (normalized counts +1)) and canonical EMT markers (Log 2 (normalized counts +1)) in the TCGA-BLCA dataset, with p -values and correlation coefficients (r) provided. f Relative YTHDC1 expression (Log 2 (normalized counts +1)) in the p-EMT high ( n = 206) and p-EMT low ( n = 206) groups in the TCGA-BLCA dataset. *** p value < 0.001.

Article Snippet: The sections were then incubated with primary YTHDC1 antibody (#29441-1-AP, Proteintech, dilution: 1:200) overnight at 4 °C.

Techniques: Comparison, Expressing, Staining

Journal: Experimental & Molecular Medicine

Article Title: Loss of YTHDC1 m 6 A reading function promotes invasiveness in urothelial carcinoma of the bladder

doi: 10.1038/s12276-024-01377-x

Figure Lengend Snippet: a Western blot showing YTHDC1 depletion (upper panel) and knockdown (lower panel) in UROtsa cells. b Viability of UROtsa cells upon YTHDC1 depletion (upper panel) or knockdown (lower panel), as analyzed by the CellTiter-Glo assay. The experiments were performed in biological and technical triplicates. p -value < 0.0001, two-way analysis of variance (ANOVA). c Colony formation assays in UROtsa YTHDC1 control (Ctrl) and YTHDC1-depleted (KO) cells (above) or UROtsa control (Scr3) and YTHDC1-knockdown (Sh3) cells (below). Representative images are displayed on the left, while the quantification and statistics of three independent replicates using the ColonyArea algorithm in ImageJ are shown on the right. *** p -value < 0.001, * p -value < 0.05; unpaired two-sided t test. d Apoptotic cell levels in UROtsa cells depleted of YTHDC1 compared with those in control (above) and sh3 vs. Scr3 cells (below), as measured by Caspase-3/7-Glo assays with biological and technical triplicates. ** p -value < 0.01, unpaired two-sided t test . e Transwell migration assays for YTHDC1 Ctrl and YTHDC1-depleted (upper panel) or Scr3 and Sh3 (lower panel) UROtsa cells. Representative images were taken using a 20x objective lens and are displayed on the left, while the quantification and statistics of the relative number of migrated cells are shown on the right. ** p -value < 0.01, * p -value < 0.05, unpaired two-sided t test. f Transwell invasion assays for YTHDC1 Ctrl and YTHDC1-depleted (above), or Scr3 and Sh3 (below) UROtsa cells. Representative images were taken using a 20x objective lens and are displayed on the left, while the quantification and statistics of relative cell invasion are shown on the right. ** p -value < 0.01, * p -value < 0.05, unpaired two-sided t test. All Transwell experiments were performed with 3 biological replicates. g Colony formation assays in BLCA cell lines (UM-UC-3, T24, RT112, and RT4) with the YTHDC1 empty vector (EV) or YTHDC1-overexpressing (OE) cells. Representative images of UM-UC-3 cells are displayed on the left, while quantification and statistics of all BLCA cell lines from three independent replicates using the ColonyArea algorithm in ImageJ are shown on the right. *** p -value < 0.001, unpaired two-sided t test. h Apoptosis levels in BLCA cell lines with YTHDC1 overexpression compared with those with empty vector, as measured by Caspase-3/7-Glo assays with biological and technical triplicates. *** p -value < 0.001, unpaired two-sided t test. i Transwell invasion assays in BLCA cell lines with YTHDC1 EV and YTHDC1 OE. The representative images shown are the invasion assays in UM-UC-3 cell lines, which were taken with a 20x objective lens and are displayed on the left. Representative images of UM-UC-3 cells (left, 20x objective) and quantification of all of the cell lines (right) are shown. *** p -value < 0.001, ** p -value < 0.01, unpaired two-sided t test. j Quantification of migration assays in BLCA cell lines with empty vector or YTHDC1 overexpression. *** p -value < 0.001, ** p -value < 0.01, unpaired two-sided t test.

Article Snippet: The sections were then incubated with primary YTHDC1 antibody (#29441-1-AP, Proteintech, dilution: 1:200) overnight at 4 °C.

Techniques: Western Blot, Knockdown, Glo Assay, Control, Migration, Plasmid Preparation, Over Expression

Journal: Experimental & Molecular Medicine

Article Title: Loss of YTHDC1 m 6 A reading function promotes invasiveness in urothelial carcinoma of the bladder

doi: 10.1038/s12276-024-01377-x

Figure Lengend Snippet: a Schematic illustration of our inhibitor approach to disrupt the interaction of YTHDC1 with m 6 A-modified RNAs, with the molecular structure of the YTHDC1 inhibitor depicted on the right. b Western blot showing the protein levels of YTHDC1 and METTL3 in wild-type UROtsa cells after treatment with the YTHDC1 inhibitor. The quantification results obtained via ImageJ are shown on the right. * p -value < 0.05; ns: not significant; unpaired two-sided t -test. c Transwell migration assays of UROtsa cells treated with DMSO (control) or the YTHDC1 inhibitor. Representative images were taken using a 20x objective lens and are displayed on the left, while the quantification and statistics of relative cell migration in three independent replicates are shown on the right. *** p -value < 0.001, unpaired two-sided t test. d Transwell invasion assays for UROtsa cells treated with DMSO (control) or the YTHDC1 inhibitor. Representative images were taken using a 20x objective lens and are displayed on the left, while the quantification and statistics of relative cell invasion in three independent replicates are shown on the right. * p -value < 0.05, unpaired two-sided t test.

Article Snippet: The sections were then incubated with primary YTHDC1 antibody (#29441-1-AP, Proteintech, dilution: 1:200) overnight at 4 °C.

Techniques: Modification, Western Blot, Migration, Control

Journal: Experimental & Molecular Medicine

Article Title: Loss of YTHDC1 m 6 A reading function promotes invasiveness in urothelial carcinoma of the bladder

doi: 10.1038/s12276-024-01377-x

Figure Lengend Snippet: a Volcano plot illustrating the significantly altered transcripts in YTHDC1-depleted cells compared with Ctrl cells. Downregulated genes are shown in blue, whereas upregulated genes are shown in red. q < 0.05. b GSEA plots demonstrating the specific dysregulation of gene sets associated with EMT and cell adhesion in YTHDC1-depleted cells compared with control UROtsa cells. The plots display the normalized enriched score (NES) and corresponding p -values. c GO analysis of the DEGs between YTHDC1-depleted and control UROtsa cells, highlighting the enrichment of metastasis-related processes. d Venn diagram showing the overlap between the differentially expressed transcripts ( | Log 2 Foldchange (FC)| > 1.5, q < 0.05) upon YTHDC1 depletion in UROtsa cells and those known to be m 6 A modified, as recently determined by GLORI mapping . e Heatmaps presenting the expression levels of overlapping transcripts displayed in ( d ), specifically focusing on the terms related to metastasis from the GO analysis. f Transcripts in ( e ) exhibiting the highest Pearson correlation with YTHDC1 expression in TCGA-BLCA samples, with p -values and correlation efficiencies (r) shown in the plots.

Article Snippet: The sections were then incubated with primary YTHDC1 antibody (#29441-1-AP, Proteintech, dilution: 1:200) overnight at 4 °C.

Techniques: Control, Modification, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Loss of YTHDC1 m 6 A reading function promotes invasiveness in urothelial carcinoma of the bladder

doi: 10.1038/s12276-024-01377-x

Figure Lengend Snippet: a Representative Western blot showing YTHDC1 protein abundance in DMSO- and STM2457-treated wild-type UROtsa cells. IgG was used as an IP control. b Read counts (upper panel) from YTHDC1 RIP-seq data in the DMSO group, demonstrating enriched YTHDC1 binding across all transcripts. Blue line: YTHDC1 pulldown; red line: input signal. Heatmap (lower panel) showing enrichment of YTHDC1-bound transcripts in the 5’ to 3’ direction. Each row corresponds to a transcript bound by YTHDC1, where the intensity of the color reflects the enrichment level, with the scale bar shown on the right. c Pie charts displaying the distribution of YTHDC1 RIP-seq peaks in the DMSO and STM2457 groups (q < 0.01). d Venn diagram indicating the number of YTHDC1 binding targets in the DMSO and STM2457 treatment groups. p -value < 0.05. e Representative genome tracks depicting an example gene, STEAP1, bound by YTHDC1, with lower peaks in the STM2457 treatment group than in the DMSO group. f Heatmaps illustrating enriched YTHDC1 binding (normalized against the input control) in the DMSO and STM2457 treatment groups for all high-confidence YTHDC1-bound transcripts (determined by Diffbind, log 2 fold change < −1, p -value < 0.05). g Upper row: enriched motifs in YTHDC1-bound sequences identified by RIP-seq in the DMSO group ( p -value = 1e-35); lower row: enriched motifs in YTHDC1-bound sequences identified by RIP-seq in the high-confidence group ( p -value = 1e-22). h Venn diagram showing the intersection among high-confidence YTHDC1-bound transcripts, m 6 A-modified transcripts, and differentially expressed transcripts in YTHDC1-depleted cells ( | log 2 fold change | > 1.5, q -value < 0.05). The intersecting genes are listed in the middle, and representative peaks (signal intensity) are shown in the right panel to demonstrate the overlap between m 6 A peaks (GLORI ) and YTHDC1 binding peaks (RIP-seq).

Article Snippet: The sections were then incubated with primary YTHDC1 antibody (#29441-1-AP, Proteintech, dilution: 1:200) overnight at 4 °C.

Techniques: Western Blot, Quantitative Proteomics, Control, Binding Assay, Modification

Journal: Experimental & Molecular Medicine

Article Title: Loss of YTHDC1 m 6 A reading function promotes invasiveness in urothelial carcinoma of the bladder

doi: 10.1038/s12276-024-01377-x

Figure Lengend Snippet: a Left panel: Pearson correlation analysis between YTHDC1 expression (Log 2 (normalized counts +1)) and SMAD6 (Log 2 (normalized counts +1)) in the TCGA-BLCA dataset, with p values and correlation coefficients (r) provided. Right panel: Ten-year overall survival analysis of SMAD6 in the TCGA-BLCA dataset. Log-rank test, p -value = 0.0001. b Comparison of SMAD6 expression in NMIBC and MIBC using data from the Fudan and UROMOL cohorts. *** p -value < 0.001, Mann‒Whitney U test. c Representative images of SMAD6 mRNA (yellow) and YTHDC1 protein (magenta) detected in paratumoral (upper row) and tumoral (lower row) FFPE tissues, respectively. The white arrows indicate SMAD6 -YTHDC1 colocalization. Scale bar = 10 μM. d Left panel: Colocalization detection results using Big-FISH of the abovementioned paratumoral and tumoral tissues. Scale bar = 10 μM. Right panel: Quantification of the percentage of colocalized SMAD6 foci. * p -value < 0.05, paired Student’s t test. e Relative luciferase activity in control and YTHDC1-depleted UROtsa cells transfected with wild-type or m 6 A-mutant SMAD6 5’UTR constructs. Three independent experiments were performed. *** p -value < 0.001, ns: not significant. f Time course qPCR analysis of SMAD6 mRNA expression in UROtsa cells following transfection with nontargeting control siRNA (siNC) or SMAD6 -targeting siRNA (siSMAD6). The experiments were performed with 3 biological replicates. *** p -value < 0.001, ** p -value < 0.01, * p -value < 0.05. g Quantification of Transwell invasion assays of UROtsa cells transfected with siNC and siSMAD6 after 24 h. Three biological replicates, ** p -value < 0.01. h Quantification of Transwell invasion assays of UROtsa Ctrl and YTHDC1-depleted cells transduced with empty vector (EV) and SMAD6 overexpression (OE). *** p -value < 0.001, ** p -value < 0.01, ns: not significant. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used.

Article Snippet: The sections were then incubated with primary YTHDC1 antibody (#29441-1-AP, Proteintech, dilution: 1:200) overnight at 4 °C.

Techniques: Expressing, Comparison, Luciferase, Activity Assay, Control, Transfection, Mutagenesis, Construct, Transduction, Plasmid Preparation, Over Expression

Journal: Journal of Inflammation (London, England)

Article Title: YTHDC1 inhibits autophagy-dependent NF-κB signaling by stabilizing Beclin1 mRNA in macrophages

doi: 10.1186/s12950-024-00393-y

Figure Lengend Snippet: Primers for qRT‒PCR

Article Snippet: The membranes were incubated with primary antibodies against YTHDC1 (1:1000, ab259990, Abcam), iNOS (1:1000, ab178945, Abcam), p-p65 (1:200, sc-136,548, Santa Cruz), p65 (1:1000, 10745-1-AP, Proteintech), SQSTM1 (1:50000, ab109012, Abcam), Beclin1 (1:500, ab210498, Abcam), LC3A/B (1:500, 4108s, CST), and β-actin (1:2000, 66009-1-Ig, Proteintech) at 4 ℃ overnight.

Techniques: Sequencing

Journal: Journal of Inflammation (London, England)

Article Title: YTHDC1 inhibits autophagy-dependent NF-κB signaling by stabilizing Beclin1 mRNA in macrophages

doi: 10.1186/s12950-024-00393-y

Figure Lengend Snippet: YTHDC1 expression in DSS-induced experimental mouse colitis. A YTHDC1 protein levels were detected by immunoblotting ( n = 6). B Statistics of YTHDC1 expression in each group. C Colocalization of YTHDC1 and macrophages in mouse colon tissue was determined by immunofluorescence staining ( n = 3). YTHDC1 and F4/80 are labeled red and green, respectively. Nuclei stained with DAPI are shown in blue. Data are the means ± SEMs. Statistical significance was determined using unpaired Student’s t test. *** p < 0.001

Article Snippet: The membranes were incubated with primary antibodies against YTHDC1 (1:1000, ab259990, Abcam), iNOS (1:1000, ab178945, Abcam), p-p65 (1:200, sc-136,548, Santa Cruz), p65 (1:1000, 10745-1-AP, Proteintech), SQSTM1 (1:50000, ab109012, Abcam), Beclin1 (1:500, ab210498, Abcam), LC3A/B (1:500, 4108s, CST), and β-actin (1:2000, 66009-1-Ig, Proteintech) at 4 ℃ overnight.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Labeling

Journal: Journal of Inflammation (London, England)

Article Title: YTHDC1 inhibits autophagy-dependent NF-κB signaling by stabilizing Beclin1 mRNA in macrophages

doi: 10.1186/s12950-024-00393-y

Figure Lengend Snippet: YTHDC1 is involved in LPS/IFN-γ-stimulated inflammation of RAW264.7 cells. A Bright-field images of RAW264.7 cells cultured without or with 100 ng/ml LPS and 10 ng/ml IFN-γ. B mRNA levels of iNOS , CD86 , IL-6 and TNF-α following LPS/IFN-γ treatment for 12 h ( n = 3). C YTHDC1 mRNA levels were assessed after LPS/IFN-γ treatment for 12 h ( n = 4). D YTHDC1 protein expression was detected by immunoblotting following LPS/IFN-γ stimulation for 24 h ( n = 3). E Quantification of the YTHDC1 band intensity. Data are the means ± SEMs. Statistical significance was determined using unpaired Student’s t test. ** p < 0.01, *** p < 0.001

Article Snippet: The membranes were incubated with primary antibodies against YTHDC1 (1:1000, ab259990, Abcam), iNOS (1:1000, ab178945, Abcam), p-p65 (1:200, sc-136,548, Santa Cruz), p65 (1:1000, 10745-1-AP, Proteintech), SQSTM1 (1:50000, ab109012, Abcam), Beclin1 (1:500, ab210498, Abcam), LC3A/B (1:500, 4108s, CST), and β-actin (1:2000, 66009-1-Ig, Proteintech) at 4 ℃ overnight.

Techniques: Cell Culture, Expressing, Western Blot

Journal: Journal of Inflammation (London, England)

Article Title: YTHDC1 inhibits autophagy-dependent NF-κB signaling by stabilizing Beclin1 mRNA in macrophages

doi: 10.1186/s12950-024-00393-y

Figure Lengend Snippet: YTHDC1 alleviated LPS/IFN-γ-induced inflammation in RAW264.7 macrophages. A Fluorescence photos of RAW264.7 cells stably overexpressing YTHDC1. B Protein expression of YTHDC1 following transfection with YTHDC1 and negative control. C WB detection of iNOS after LPS/IFN-γ treatment for 24 h ( n = 3). D Quantification of the iNOS band intensity. E Phosphorylation of p65 was detected following LPS/IFN-γ treatment for 30 min ( n = 3). F Quantification of the p-p65 band intensity. G iNOS , CD86 , IL-6 and TNF-α mRNA levels in YTHDC1-overexpressing macrophages after LPS/IFN-γ treatment for 12 h ( n = 3). H Cells were pretreated with 10 µM JSH-23 for 2 h followed by costimulation with LPS/IFN-γ for 12 h. CD86 and IL-6 mRNA levels were assayed by qPCR ( n = 3). Data are the means ± SEMs. Statistical significance was determined using one-way or two-way ANOVA with Bonferroni’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001. NS, no significance

Article Snippet: The membranes were incubated with primary antibodies against YTHDC1 (1:1000, ab259990, Abcam), iNOS (1:1000, ab178945, Abcam), p-p65 (1:200, sc-136,548, Santa Cruz), p65 (1:1000, 10745-1-AP, Proteintech), SQSTM1 (1:50000, ab109012, Abcam), Beclin1 (1:500, ab210498, Abcam), LC3A/B (1:500, 4108s, CST), and β-actin (1:2000, 66009-1-Ig, Proteintech) at 4 ℃ overnight.

Techniques: Fluorescence, Stable Transfection, Expressing, Transfection, Negative Control

Journal: Journal of Inflammation (London, England)

Article Title: YTHDC1 inhibits autophagy-dependent NF-κB signaling by stabilizing Beclin1 mRNA in macrophages

doi: 10.1186/s12950-024-00393-y

Figure Lengend Snippet: YTHDC1 regulated autophagy-dependent NF-κB signaling. A WB analysis of YTHDC1 in shNC- and shYTHDC1-transfected RAW264.7 macrophages ( n = 3). B Quantification of the YTHDC1 band intensity. C iNOS protein levels in YTHDC1-silenced macrophages induced by LPS/IFN-γ for 24 h ( n = 4). D Quantification of the iNOS band intensity. E SQSTM1, Beclin1, and LC3 expression detected by western blot in YTHDC1-knockdown macrophages induced by LPS/IFN-γ for 24 h ( n = 3–5). F Quantification of the band intensity of SQSTM1, Beclin1, LC3II and YTHDC1. G - I qPCR and WB analysis of Beclin1 in shNC- and shBeclin1-transfected RAW264.7 macrophages ( n = 3). J Phosphorylation of p65 was detected in LV-YTHDC1- and shBeclin1-cotransfected RAW264.7 cells after LPS/IFN-γ treatment for 30 min ( n = 3). K Quantification of the p-p65 band intensity. Data are the means ± SEMs. Statistical significance was determined using unpaired Student’s t test and one-way or two-way ANOVA with Bonferroni’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.05

Article Snippet: The membranes were incubated with primary antibodies against YTHDC1 (1:1000, ab259990, Abcam), iNOS (1:1000, ab178945, Abcam), p-p65 (1:200, sc-136,548, Santa Cruz), p65 (1:1000, 10745-1-AP, Proteintech), SQSTM1 (1:50000, ab109012, Abcam), Beclin1 (1:500, ab210498, Abcam), LC3A/B (1:500, 4108s, CST), and β-actin (1:2000, 66009-1-Ig, Proteintech) at 4 ℃ overnight.

Techniques: Transfection, Expressing, Western Blot

Journal: Journal of Inflammation (London, England)

Article Title: YTHDC1 inhibits autophagy-dependent NF-κB signaling by stabilizing Beclin1 mRNA in macrophages

doi: 10.1186/s12950-024-00393-y

Figure Lengend Snippet: YTHDC1 stabilized Beclin1 mRNA. A Half-life of Beclin1 mRNA in YTHDC1-knockdown RAW264.7 cells followed by actinomycin D treatment for the indicated times ( n = 3). B Half-life of Beclin1 mRNA in YTHDC1-overexpressing RAW264.7 cells followed by actinomycin D treatment for the indicated times ( n = 3). C RIP-qPCR detected the binding of YTHDC1 to Beclin1 mRNA ( n = 3). D MeRIP-qPCR confirmed m(6)A modification of Beclin1 transcript ( n = 3). Data are the means ± SEMs. Statistical significance was determined using unpaired Student’s t test and two-way ANOVA with Bonferroni’s post hoc test. * p < 0.05, ** p < 0.01

Article Snippet: The membranes were incubated with primary antibodies against YTHDC1 (1:1000, ab259990, Abcam), iNOS (1:1000, ab178945, Abcam), p-p65 (1:200, sc-136,548, Santa Cruz), p65 (1:1000, 10745-1-AP, Proteintech), SQSTM1 (1:50000, ab109012, Abcam), Beclin1 (1:500, ab210498, Abcam), LC3A/B (1:500, 4108s, CST), and β-actin (1:2000, 66009-1-Ig, Proteintech) at 4 ℃ overnight.

Techniques: Binding Assay, Modification

Journal: Journal of Inflammation (London, England)

Article Title: YTHDC1 inhibits autophagy-dependent NF-κB signaling by stabilizing Beclin1 mRNA in macrophages

doi: 10.1186/s12950-024-00393-y

Figure Lengend Snippet: >Schematic representation for the role of YTHDC1 in response to LPS/IFN-γ stimulation in macrophages. The figure was drawn by Figdraw

Article Snippet: The membranes were incubated with primary antibodies against YTHDC1 (1:1000, ab259990, Abcam), iNOS (1:1000, ab178945, Abcam), p-p65 (1:200, sc-136,548, Santa Cruz), p65 (1:1000, 10745-1-AP, Proteintech), SQSTM1 (1:50000, ab109012, Abcam), Beclin1 (1:500, ab210498, Abcam), LC3A/B (1:500, 4108s, CST), and β-actin (1:2000, 66009-1-Ig, Proteintech) at 4 ℃ overnight.

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway

doi: 10.1186/s13046-022-02460-9

Figure Lengend Snippet: The downregulation of YTHDC1 is associated with the poor prognosis in ccRCC. A , The expression level of YTHDC1 was analyzed by the ENCORI web tool in various types of solid tumor. P values and HR were indicated in the panel. B , The expression level of YTHDC1 in KIRC was analyzed by the Timer 2.0 ( http://timer.cistrome.org/ ). *, P < 0.05. C , The prognosis of YTHDC1 in KIRC was determined by the ENCORI web tool. P values as indicated. D , CancerSEA web tool was used to analyze the biological function of YTHDC1 in the renal cell carcinoma. E-I , A498 and 786-O cells were transfected with indicated shRNAs for 72 h. After puromycin selection, cells were harvested for western blot analysis ( E ), RT-qPCR assay ( F ), CCK-8 assay ( G ), wound healing assay ( H ), transwell assay ( I ). Data presents as mean ± SD with three replicates. ***, P < 0.001. J-N , A498 and 786-O cells were transfected with indicated plasmids for 24 h. Cells were harvested for western blot analysis ( J ), RT-qPCR assay ( K ), CCK-8 assay ( L ), wound healing assay ( M ), transwell assay ( N ). Data presents as mean ± SD with three replicates. ***, P < 0.001

Article Snippet: The primary antibodies used were as follows: YTHDC1 (#29441-1-AP, Proteintech, 1:500 dilution), ANXA1 (#55018-1-AP, Proteintech, 1:2000 dilution), YY1 (#66281-1-Ig, Proteintech, 1:1000 dilution), HDAC2 (#12922-3-AP, Proteintech, 1:1000 dilution), and beta actin (#66009-1-Ig, Proteintech, 1:5000 dilution).

Techniques: Expressing, Transfection, Selection, Western Blot, Quantitative RT-PCR, CCK-8 Assay, Wound Healing Assay, Transwell Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway

doi: 10.1186/s13046-022-02460-9

Figure Lengend Snippet: Knockdown of YTHDC1 promotes the progression of ccRCC in vivo . A-E , 786-O cells were transfected with indicated shRNAs for 72 h. After puromycin selection, cells were subcutaneously injected into the nude mice. The tumor image was shown in panel A , the tumor mass was shown in panel B , the tumor growth curve was shown in panel C . The excised tumors were subjected to IHC staining of YTHDC1 ( D ) or Ki-67 ( E ). Data presents as mean ± SD with six replicates. ***, P < 0.001. F-J , A498 cells were transfected with indicated shRNAs for 72 h. After puromycin selection, cells were subcutaneously injected into the nude mice. The tumor image was shown in panel F, the tumor mass was shown in panel G , the tumor growth curve was shown in panel H . The excised tumors were subjected to IHC staining of YTHDC1 ( I ) or Ki-67 ( J ). Data presents as mean ± SD with four replicates. **, P < 0.01; ***, P < 0.001

Article Snippet: The primary antibodies used were as follows: YTHDC1 (#29441-1-AP, Proteintech, 1:500 dilution), ANXA1 (#55018-1-AP, Proteintech, 1:2000 dilution), YY1 (#66281-1-Ig, Proteintech, 1:1000 dilution), HDAC2 (#12922-3-AP, Proteintech, 1:1000 dilution), and beta actin (#66009-1-Ig, Proteintech, 1:5000 dilution).

Techniques: Knockdown, In Vivo, Transfection, Selection, Injection, Immunohistochemistry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway

doi: 10.1186/s13046-022-02460-9

Figure Lengend Snippet: YTHDC1 contributes to the inactivation of the ERK/MAPK signaling pathway in ccRCC cells. A and B , the Reactome enrichment analysis ( A ) and KEGG enrichment analysis ( B ) of the RNA-seq of YTHDC1 in A498 cells. P values as indicated. C and D , GSEA analysis and KEGG enrichment analysis of the TCGA-KIRC dataset. E , 786-O and A498 cells were transfected with the indicates shRNAs for 72 h. After puromycin selection, cells were harvested for western blot analysis. F , 786-O and A498 cells were transfected with the indicated plasmids for 24 h. Cells were harvested for western blot analysis. G , 786-O and A498 cells were transfected with the indicates shRNAs for 72 h. Then, cells were treated with or without LY3214996 (2 µM) and subjected to CCK-8 assay. H , 86-O and A498 cells were transfected with the indicates plasmids for 24 h. Then, cells were treated with or without LY3214996 (2 µM) and subjected to CCK-8 assay. I , 786-O and A498 cells were transfected with the indicates shRNAs for 72 h. Then, cells were treated with or without GSK1120212 (20 nM) and subjected to CCK-8 assay. J , 86-O and A498 cells were transfected with the indicates plasmids for 24 h. Then, cells were treated with or without GSK1120212 (20 nM) and subjected to CCK-8 assay

Article Snippet: The primary antibodies used were as follows: YTHDC1 (#29441-1-AP, Proteintech, 1:500 dilution), ANXA1 (#55018-1-AP, Proteintech, 1:2000 dilution), YY1 (#66281-1-Ig, Proteintech, 1:1000 dilution), HDAC2 (#12922-3-AP, Proteintech, 1:1000 dilution), and beta actin (#66009-1-Ig, Proteintech, 1:5000 dilution).

Techniques: RNA Sequencing, Transfection, Selection, Western Blot, CCK-8 Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway

doi: 10.1186/s13046-022-02460-9

Figure Lengend Snippet: YTHDC1 represses ANXA1 expression in ccRCC cells. A and B , 786-O and A498 cells were transfected with the indicates shRANs for 72 h. Cell were harvested for western blot analysis and RT-qPCR analysis. Data presents as mean ± SD with four replicates. **, P < 0.01; ***, P < 0.001. C and D , 786-O and A498 cells were transfected with the indicates plasmids for 24 h. Cell were harvested for western blot analysis and RT-qPCR analysis. Data presents as mean ± SD with four replicates. **, P < 0.01; ***, P < 0.001. E , The IgG or YTHDC1 antibodies was used to performed the ChIP-qPCR assay in 786-O and A498 cells. Data presents as mean ± SD with four replicates. ***, P < 0.001. F , Magna m 6 A MeRIP kit was used to performed MeRIP-qPCR assay in 786-O and A498 cells. Data presents as mean ± SD with four replicates. **, P < 0.001. G , 786-O and A498 cells were transfected with indicated shRNAs for 72 h. Cells were harvested, and the RNA was extracted from the cytoplasm (cyto) or nucleus (mucl) respectively. The RT-qPCR assay was used to detect the mRNA of ANXA1 in 786-O and A498 cell. Data presents as mean ± SD with four replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001. H , 786-O and A498 cells were transfected with indicated plasmids for 24 h. Cells were harvested, and the RNA was extracted from the cytoplasm (cyto) or nucleus (mucl) respectively. The RT-qPCR assay was used to detect the mRNA of ANXA1 in 786-O and A498 cell. Data presents as mean ± SD with four replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001. I , 786-O and A498 cells were transfected with indicated shRNAs for 72 h. Then, cells treated with actinomycin D (5 µg/mL). Then, cells were collected at the different time points. Total RNAs were extracted and analyzed by RT-qPCR. The mRNA expression for each group was normalized to β-actin. J , 786-O and A498 cells were transfected with indicated plasmids for 72 h. Then, cells treated with actinomycin D (5 µg/mL). Then, cells were collected at the different time points. Total RNAs were extracted and analyzed by RT-qPCR. The mRNA expression for each group was normalized to β-actin. K and L , The IHC staining was performed in the tissue microarray of renal cancer by using the YTHDC1 and ANXA1 antibodies. The typical image was shown in panel K . The correlation between ANXA1 and YTHDC1 was shown in panel L , P = 0.0027

Article Snippet: The primary antibodies used were as follows: YTHDC1 (#29441-1-AP, Proteintech, 1:500 dilution), ANXA1 (#55018-1-AP, Proteintech, 1:2000 dilution), YY1 (#66281-1-Ig, Proteintech, 1:1000 dilution), HDAC2 (#12922-3-AP, Proteintech, 1:1000 dilution), and beta actin (#66009-1-Ig, Proteintech, 1:5000 dilution).

Techniques: Expressing, Transfection, Western Blot, Quantitative RT-PCR, ChIP-qPCR, Immunohistochemistry, Microarray

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway

doi: 10.1186/s13046-022-02460-9

Figure Lengend Snippet: YTHDC1 inhibits the activation of the MAPK signaling pathway by targeting ANXA1 in ccRCC. A-D , The 786-O and A498 cells were transfected with indicated constructs for 48 h. Cells were harvested for western blot analysis ( A ), CCK-8 assay ( B ), transwell assay ( C and D ). Data presents as mean ± SD with three replicates. Ns, not significant; *, P < 0.05; ***, P < 0.001. E-I , The 786-O and A498 cells were transfected with indicated shRNAs for 72 h. After puromycin selection, cells were harvested for western blot analysis ( E ), CCK-8 assay ( F ), tranwell assay ( G and H ) and colonformation assay. Data presents as mean ± SD with three replicates. Ns, not significant; *, P < 0.05; ***, P < 0.001. J and K , 786-O cells were transfected with indicated shRNAs for 72 h. After puromycin selection, cells were collected and subcutaneously injected into the nude mice. The tumor mass and tumor growth curve were shown in panel J and panel K . Data presents as mean ± SD with six replicates. Ns, not significant; *, P < 0.05; ***, P < 0.001

Article Snippet: The primary antibodies used were as follows: YTHDC1 (#29441-1-AP, Proteintech, 1:500 dilution), ANXA1 (#55018-1-AP, Proteintech, 1:2000 dilution), YY1 (#66281-1-Ig, Proteintech, 1:1000 dilution), HDAC2 (#12922-3-AP, Proteintech, 1:1000 dilution), and beta actin (#66009-1-Ig, Proteintech, 1:5000 dilution).

Techniques: Activation Assay, Transfection, Construct, Western Blot, CCK-8 Assay, Transwell Assay, Selection, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway

doi: 10.1186/s13046-022-02460-9

Figure Lengend Snippet: The YTHDC1-ANAX1 axis regulates the sensitivity of sunitinib in ccRCC. A , A498 and 786-O cells were transfected with indicated shRNAs for 72 h. Cells were treated with a serial dose of sunitinib for 24 h and harvested for CCK-8 assay. B , A498, 786-O and 786-O R (sunitinib resistance) cells were transfected with indicated plasmids for 72 h. Cells were treated with a serial dose of sunitinib for 24 h. Cells were harvested for CCK-8 assay. C and D , 786-O and A498 cells were transfected with indicated shRNAs for 72 h. Cells were treated with or without sunitinib (2 µM) and subjected to CCK-8 assay ( C ) and Annexin V-PI assay ( D ). Data presents as mean ± SD with three replicates. *, P < 0.05; ***, P < 0.001. E and F , 786-O and A498 cells were transfected with indicated plasmids for 24 h. Cells were treated with or without sunitinib (2 µM) and subjected to CCK-8 assay ( E ) and Annexin V-PI assay ( F ). Data presents as mean ± SD with three replicates. **, P < 0.01; ***, P < 0.001. G , A498 and 786-O cells were transfected with indicated shRNAs for 72 h. Cells were treated with a serial dose of sunitinib for 24 h and harvested for CCK-8 assay. H , A498 and 786-O cells were transfected with indicated constructs for 48 h. Cells were treated with a serial dose of sunitinib for 24 h and harvested for CCK-8 assay. I , A498 and 786-O cells were transfected with indicated constructs for 48 h. Cells were harvested for CCK-8 assay. Data presents as mean ± SD with three replicates. ***, P < 0.001. J , A498 and 786-O cells were transfected with indicated constructs for 72 h. Cells were harvested for CCK-8 assay. Data presents as mean ± SD with three replicates. *, P < 0.05; ***, P < 0.001. K-M , 786-O cells were transfected with indicated shRNAs for 72 h. After puromycin selection, cells were collected and subcutaneously injected into the nude mice. These mice were treated with or without sunitinib. The tumor mass and tumor growth curve were shown in panel L and panel M . Data presents as mean ± SD with six replicates. **, P < 0.05; ***, P < 0.001

Article Snippet: The primary antibodies used were as follows: YTHDC1 (#29441-1-AP, Proteintech, 1:500 dilution), ANXA1 (#55018-1-AP, Proteintech, 1:2000 dilution), YY1 (#66281-1-Ig, Proteintech, 1:1000 dilution), HDAC2 (#12922-3-AP, Proteintech, 1:1000 dilution), and beta actin (#66009-1-Ig, Proteintech, 1:5000 dilution).

Techniques: Transfection, CCK-8 Assay, Construct, Selection, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway

doi: 10.1186/s13046-022-02460-9

Figure Lengend Snippet: The YY1/HDAC2 complex downregulates the expression of YTHDC1 in ccRCC. A , The PROMO web tool predicted the potential transcriptional factors of YTHDC1. B , The Ominer web tool predicted the potential transcriptional factors of YTHDC1. C-E , 786-O and A498 cells were transfected with indicated siRNAs for 48 h. Cells were collected for western blot analysis ( C ), RT-qPCR assay ( D ), and ChIP-qPCR assay ( E ). Data presents as mean ± SD with three replicates. ***, P < 0.001. F , a diagram demonstrated the sequence and position of the YY1 binding peak in the YTHDC1 promoter. TSS transcriptional start site, WT wild type, MUT mutant type. G , 786-O and A498 cells were transfected with empty vector, GV592-YTHDC1 plasmids WT, MUT1, or MUT2 for 48 h. Cells were harvested and the activity of YTHDC1 promoter was measured. Data present as mean ± SD with three replicates. Ns, not significant; **, P < 0.01; *** P < 0.001. H , 786-O cells were transfected with indicated siRNAs for 24 h. Then, cells were transfected with EV, GV592-YTHDC1 plasmids WT another 24 h. Cells were harvested and the activity of YTHDC1 promoter was measured. Data present as mean ± SD with three replicates. Ns, not significant; *** P < 0.001. J and K , 786-O and A498 cells were transfected with indicated siRNAs for 48 h. Cells were collected for western blot analysis ( J ) and RT-qPCR assay ( K ). Data presents as mean ± SD with three replicates. ***, P < 0.001. L and M , 786-O and A498 cells were transfected with empty vector, 1 ng HDAC2 plasmids, or 5 ng HDAC2 plasmids for 24 h. Cells were harvested for western blot analysis ( L ) and RT-qPCR assay ( M ). Data presents as mean ± SD with three replicates. ***, P < 0.001. N . the ChIP-qPCR was performed by using the IgG or HDAC2 antibodies in 786-O and A498 cells. Data presents as mean ± SD with three replicates. ***, P < 0.001. O , The ChIP was firstly performed by using the YY1 antibodies. Then, the ChIP-re-ChIP assay was performed by using the IgG or HDAC2 antibodies in 786-O and A498 cells. Data presents as mean ± SD with three replicates. ***, P < 0.001. P , 786-O and A498 cells were transfected with the indicated siRNAs for 48 h. Cells were collected for western blot analysis

Article Snippet: The primary antibodies used were as follows: YTHDC1 (#29441-1-AP, Proteintech, 1:500 dilution), ANXA1 (#55018-1-AP, Proteintech, 1:2000 dilution), YY1 (#66281-1-Ig, Proteintech, 1:1000 dilution), HDAC2 (#12922-3-AP, Proteintech, 1:1000 dilution), and beta actin (#66009-1-Ig, Proteintech, 1:5000 dilution).

Techniques: Expressing, Transfection, Western Blot, Quantitative RT-PCR, ChIP-qPCR, Sequencing, Binding Assay, Mutagenesis, Plasmid Preparation, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway

doi: 10.1186/s13046-022-02460-9

Figure Lengend Snippet: HDAC2 inhibitors enhance the sensitivity of ccRCC to sunitinib. A and B , 786-O and A498 cells were treated with DMSO or CAY10683 (5 µM) for 24 h. Cells were collected for western blot analysis ( A ) and RT-qPCR assay ( B ). Data presents as mean ± SD with three replicates. ***, P < 0.001. C and D , 786-O and A498 cells were transfected with indicated plasmids for 24 h. Then, these cells were treated with DMSO or CAY10683 (5 µM) for another 24 h. Cells were collected for western blot analysis ( C ) and RT-qPCR assay ( D ). Data presents as mean ± SD with three replicates. Ns, not significant; **, P < 0.01; ***, P < 0.001. E and F , 786-O and A498 cells were transfected with indicated siRNAs for 24 h. Then, these cells were treated with DMSO or CAY10683 (5 µM) for another 24 h. Cells were collected for western blot analysis ( C ) and RT-qPCR assay ( D ). Data presents as mean ± SD with three replicates. Ns, not significant; **, P < 0.01; ***, P < 0.001. G , A498 and 786-O cells were transfected with indicated shRNAs for 48 h. Then, these cells were treated with vehicle (DMSO) or CAY10683 (5 µM) for another 24 h. These cells were harvested and treated with a serial dose of sunitinib. These cells were subjected to CCK-8 assay. H , 786-O cells were transfected with indicated shRNAs for 48 h. Then, these cells were treated with vehicle (DMSO) or CAY10683 (5 µM) for another 24 h. These cells were subjected to CCK-8 assay. Data presents as mean ± SD with three replicates. Ns, not significant; **, P < 0.01; ***, P < 0.001. I and J , 786-O cells were transfected with indicated shRNAs for 72 h. After puromycin selection, cells were collected and subcutaneously injected into the nude mice. The tumor mass was shown in panel I, and tumor growth curve was shown in panel J. Data presents as mean ± SD with six replicates. Ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. K , a model depicting that the HDAC2/YY1 complex transcriptionally represses the expression of YTHDC1, which prevents the downregulation of ANXA1 induced by YTHDC1 and activates the MAPK pathway to decrease the sensitivity of ccRCC to sunitinib. HDAC2 inhibitors treatment blocks the HDAC2/YY1/YTHDC1/ANXA1/MAPK axis to enhance the anti-tumor effect of sunitinib

Article Snippet: The primary antibodies used were as follows: YTHDC1 (#29441-1-AP, Proteintech, 1:500 dilution), ANXA1 (#55018-1-AP, Proteintech, 1:2000 dilution), YY1 (#66281-1-Ig, Proteintech, 1:1000 dilution), HDAC2 (#12922-3-AP, Proteintech, 1:1000 dilution), and beta actin (#66009-1-Ig, Proteintech, 1:5000 dilution).

Techniques: Western Blot, Quantitative RT-PCR, Transfection, CCK-8 Assay, Selection, Injection, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway

doi: 10.1186/s13046-022-02460-9

Figure Lengend Snippet: The downregulation of YTHDC1 is associated with the poor prognosis in ccRCC. A , The expression level of YTHDC1 was analyzed by the ENCORI web tool in various types of solid tumor. P values and HR were indicated in the panel. B , The expression level of YTHDC1 in KIRC was analyzed by the Timer 2.0 ( http://timer.cistrome.org/ ). *, P < 0.05. C , The prognosis of YTHDC1 in KIRC was determined by the ENCORI web tool. P values as indicated. D , CancerSEA web tool was used to analyze the biological function of YTHDC1 in the renal cell carcinoma. E-I , A498 and 786-O cells were transfected with indicated shRNAs for 72 h. After puromycin selection, cells were harvested for western blot analysis ( E ), RT-qPCR assay ( F ), CCK-8 assay ( G ), wound healing assay ( H ), transwell assay ( I ). Data presents as mean ± SD with three replicates. ***, P < 0.001. J-N , A498 and 786-O cells were transfected with indicated plasmids for 24 h. Cells were harvested for western blot analysis ( J ), RT-qPCR assay ( K ), CCK-8 assay ( L ), wound healing assay ( M ), transwell assay ( N ). Data presents as mean ± SD with three replicates. ***, P < 0.001

Article Snippet: Immunohistochemistry was performed with primary antibodies against YTHDC1 (#29441-1-AP, Proteintech, 1:1000, ANXA1 (21990-1-AP, Proteintech; 1:10000 dilution) and TMA slides (#KD1921, Avilabio, China).

Techniques: Expressing, Transfection, Selection, Western Blot, Quantitative RT-PCR, CCK-8 Assay, Wound Healing Assay, Transwell Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway

doi: 10.1186/s13046-022-02460-9

Figure Lengend Snippet: Knockdown of YTHDC1 promotes the progression of ccRCC in vivo . A-E , 786-O cells were transfected with indicated shRNAs for 72 h. After puromycin selection, cells were subcutaneously injected into the nude mice. The tumor image was shown in panel A , the tumor mass was shown in panel B , the tumor growth curve was shown in panel C . The excised tumors were subjected to IHC staining of YTHDC1 ( D ) or Ki-67 ( E ). Data presents as mean ± SD with six replicates. ***, P < 0.001. F-J , A498 cells were transfected with indicated shRNAs for 72 h. After puromycin selection, cells were subcutaneously injected into the nude mice. The tumor image was shown in panel F, the tumor mass was shown in panel G , the tumor growth curve was shown in panel H . The excised tumors were subjected to IHC staining of YTHDC1 ( I ) or Ki-67 ( J ). Data presents as mean ± SD with four replicates. **, P < 0.01; ***, P < 0.001

Article Snippet: Immunohistochemistry was performed with primary antibodies against YTHDC1 (#29441-1-AP, Proteintech, 1:1000, ANXA1 (21990-1-AP, Proteintech; 1:10000 dilution) and TMA slides (#KD1921, Avilabio, China).

Techniques: Knockdown, In Vivo, Transfection, Selection, Injection, Immunohistochemistry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway

doi: 10.1186/s13046-022-02460-9

Figure Lengend Snippet: YTHDC1 contributes to the inactivation of the ERK/MAPK signaling pathway in ccRCC cells. A and B , the Reactome enrichment analysis ( A ) and KEGG enrichment analysis ( B ) of the RNA-seq of YTHDC1 in A498 cells. P values as indicated. C and D , GSEA analysis and KEGG enrichment analysis of the TCGA-KIRC dataset. E , 786-O and A498 cells were transfected with the indicates shRNAs for 72 h. After puromycin selection, cells were harvested for western blot analysis. F , 786-O and A498 cells were transfected with the indicated plasmids for 24 h. Cells were harvested for western blot analysis. G , 786-O and A498 cells were transfected with the indicates shRNAs for 72 h. Then, cells were treated with or without LY3214996 (2 µM) and subjected to CCK-8 assay. H , 86-O and A498 cells were transfected with the indicates plasmids for 24 h. Then, cells were treated with or without LY3214996 (2 µM) and subjected to CCK-8 assay. I , 786-O and A498 cells were transfected with the indicates shRNAs for 72 h. Then, cells were treated with or without GSK1120212 (20 nM) and subjected to CCK-8 assay. J , 86-O and A498 cells were transfected with the indicates plasmids for 24 h. Then, cells were treated with or without GSK1120212 (20 nM) and subjected to CCK-8 assay

Article Snippet: Immunohistochemistry was performed with primary antibodies against YTHDC1 (#29441-1-AP, Proteintech, 1:1000, ANXA1 (21990-1-AP, Proteintech; 1:10000 dilution) and TMA slides (#KD1921, Avilabio, China).

Techniques: RNA Sequencing, Transfection, Selection, Western Blot, CCK-8 Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway

doi: 10.1186/s13046-022-02460-9

Figure Lengend Snippet: YTHDC1 represses ANXA1 expression in ccRCC cells. A and B , 786-O and A498 cells were transfected with the indicates shRANs for 72 h. Cell were harvested for western blot analysis and RT-qPCR analysis. Data presents as mean ± SD with four replicates. **, P < 0.01; ***, P < 0.001. C and D , 786-O and A498 cells were transfected with the indicates plasmids for 24 h. Cell were harvested for western blot analysis and RT-qPCR analysis. Data presents as mean ± SD with four replicates. **, P < 0.01; ***, P < 0.001. E , The IgG or YTHDC1 antibodies was used to performed the ChIP-qPCR assay in 786-O and A498 cells. Data presents as mean ± SD with four replicates. ***, P < 0.001. F , Magna m 6 A MeRIP kit was used to performed MeRIP-qPCR assay in 786-O and A498 cells. Data presents as mean ± SD with four replicates. **, P < 0.001. G , 786-O and A498 cells were transfected with indicated shRNAs for 72 h. Cells were harvested, and the RNA was extracted from the cytoplasm (cyto) or nucleus (mucl) respectively. The RT-qPCR assay was used to detect the mRNA of ANXA1 in 786-O and A498 cell. Data presents as mean ± SD with four replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001. H , 786-O and A498 cells were transfected with indicated plasmids for 24 h. Cells were harvested, and the RNA was extracted from the cytoplasm (cyto) or nucleus (mucl) respectively. The RT-qPCR assay was used to detect the mRNA of ANXA1 in 786-O and A498 cell. Data presents as mean ± SD with four replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001. I , 786-O and A498 cells were transfected with indicated shRNAs for 72 h. Then, cells treated with actinomycin D (5 µg/mL). Then, cells were collected at the different time points. Total RNAs were extracted and analyzed by RT-qPCR. The mRNA expression for each group was normalized to β-actin. J , 786-O and A498 cells were transfected with indicated plasmids for 72 h. Then, cells treated with actinomycin D (5 µg/mL). Then, cells were collected at the different time points. Total RNAs were extracted and analyzed by RT-qPCR. The mRNA expression for each group was normalized to β-actin. K and L , The IHC staining was performed in the tissue microarray of renal cancer by using the YTHDC1 and ANXA1 antibodies. The typical image was shown in panel K . The correlation between ANXA1 and YTHDC1 was shown in panel L , P = 0.0027

Article Snippet: Immunohistochemistry was performed with primary antibodies against YTHDC1 (#29441-1-AP, Proteintech, 1:1000, ANXA1 (21990-1-AP, Proteintech; 1:10000 dilution) and TMA slides (#KD1921, Avilabio, China).

Techniques: Expressing, Transfection, Western Blot, Quantitative RT-PCR, ChIP-qPCR, Immunohistochemistry, Microarray

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway

doi: 10.1186/s13046-022-02460-9

Figure Lengend Snippet: YTHDC1 inhibits the activation of the MAPK signaling pathway by targeting ANXA1 in ccRCC. A-D , The 786-O and A498 cells were transfected with indicated constructs for 48 h. Cells were harvested for western blot analysis ( A ), CCK-8 assay ( B ), transwell assay ( C and D ). Data presents as mean ± SD with three replicates. Ns, not significant; *, P < 0.05; ***, P < 0.001. E-I , The 786-O and A498 cells were transfected with indicated shRNAs for 72 h. After puromycin selection, cells were harvested for western blot analysis ( E ), CCK-8 assay ( F ), tranwell assay ( G and H ) and colonformation assay. Data presents as mean ± SD with three replicates. Ns, not significant; *, P < 0.05; ***, P < 0.001. J and K , 786-O cells were transfected with indicated shRNAs for 72 h. After puromycin selection, cells were collected and subcutaneously injected into the nude mice. The tumor mass and tumor growth curve were shown in panel J and panel K . Data presents as mean ± SD with six replicates. Ns, not significant; *, P < 0.05; ***, P < 0.001

Article Snippet: Immunohistochemistry was performed with primary antibodies against YTHDC1 (#29441-1-AP, Proteintech, 1:1000, ANXA1 (21990-1-AP, Proteintech; 1:10000 dilution) and TMA slides (#KD1921, Avilabio, China).

Techniques: Activation Assay, Transfection, Construct, Western Blot, CCK-8 Assay, Transwell Assay, Selection, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway

doi: 10.1186/s13046-022-02460-9

Figure Lengend Snippet: The YTHDC1-ANAX1 axis regulates the sensitivity of sunitinib in ccRCC. A , A498 and 786-O cells were transfected with indicated shRNAs for 72 h. Cells were treated with a serial dose of sunitinib for 24 h and harvested for CCK-8 assay. B , A498, 786-O and 786-O R (sunitinib resistance) cells were transfected with indicated plasmids for 72 h. Cells were treated with a serial dose of sunitinib for 24 h. Cells were harvested for CCK-8 assay. C and D , 786-O and A498 cells were transfected with indicated shRNAs for 72 h. Cells were treated with or without sunitinib (2 µM) and subjected to CCK-8 assay ( C ) and Annexin V-PI assay ( D ). Data presents as mean ± SD with three replicates. *, P < 0.05; ***, P < 0.001. E and F , 786-O and A498 cells were transfected with indicated plasmids for 24 h. Cells were treated with or without sunitinib (2 µM) and subjected to CCK-8 assay ( E ) and Annexin V-PI assay ( F ). Data presents as mean ± SD with three replicates. **, P < 0.01; ***, P < 0.001. G , A498 and 786-O cells were transfected with indicated shRNAs for 72 h. Cells were treated with a serial dose of sunitinib for 24 h and harvested for CCK-8 assay. H , A498 and 786-O cells were transfected with indicated constructs for 48 h. Cells were treated with a serial dose of sunitinib for 24 h and harvested for CCK-8 assay. I , A498 and 786-O cells were transfected with indicated constructs for 48 h. Cells were harvested for CCK-8 assay. Data presents as mean ± SD with three replicates. ***, P < 0.001. J , A498 and 786-O cells were transfected with indicated constructs for 72 h. Cells were harvested for CCK-8 assay. Data presents as mean ± SD with three replicates. *, P < 0.05; ***, P < 0.001. K-M , 786-O cells were transfected with indicated shRNAs for 72 h. After puromycin selection, cells were collected and subcutaneously injected into the nude mice. These mice were treated with or without sunitinib. The tumor mass and tumor growth curve were shown in panel L and panel M . Data presents as mean ± SD with six replicates. **, P < 0.05; ***, P < 0.001

Article Snippet: Immunohistochemistry was performed with primary antibodies against YTHDC1 (#29441-1-AP, Proteintech, 1:1000, ANXA1 (21990-1-AP, Proteintech; 1:10000 dilution) and TMA slides (#KD1921, Avilabio, China).

Techniques: Transfection, CCK-8 Assay, Construct, Selection, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway

doi: 10.1186/s13046-022-02460-9

Figure Lengend Snippet: The YY1/HDAC2 complex downregulates the expression of YTHDC1 in ccRCC. A , The PROMO web tool predicted the potential transcriptional factors of YTHDC1. B , The Ominer web tool predicted the potential transcriptional factors of YTHDC1. C-E , 786-O and A498 cells were transfected with indicated siRNAs for 48 h. Cells were collected for western blot analysis ( C ), RT-qPCR assay ( D ), and ChIP-qPCR assay ( E ). Data presents as mean ± SD with three replicates. ***, P < 0.001. F , a diagram demonstrated the sequence and position of the YY1 binding peak in the YTHDC1 promoter. TSS transcriptional start site, WT wild type, MUT mutant type. G , 786-O and A498 cells were transfected with empty vector, GV592-YTHDC1 plasmids WT, MUT1, or MUT2 for 48 h. Cells were harvested and the activity of YTHDC1 promoter was measured. Data present as mean ± SD with three replicates. Ns, not significant; **, P < 0.01; *** P < 0.001. H , 786-O cells were transfected with indicated siRNAs for 24 h. Then, cells were transfected with EV, GV592-YTHDC1 plasmids WT another 24 h. Cells were harvested and the activity of YTHDC1 promoter was measured. Data present as mean ± SD with three replicates. Ns, not significant; *** P < 0.001. J and K , 786-O and A498 cells were transfected with indicated siRNAs for 48 h. Cells were collected for western blot analysis ( J ) and RT-qPCR assay ( K ). Data presents as mean ± SD with three replicates. ***, P < 0.001. L and M , 786-O and A498 cells were transfected with empty vector, 1 ng HDAC2 plasmids, or 5 ng HDAC2 plasmids for 24 h. Cells were harvested for western blot analysis ( L ) and RT-qPCR assay ( M ). Data presents as mean ± SD with three replicates. ***, P < 0.001. N . the ChIP-qPCR was performed by using the IgG or HDAC2 antibodies in 786-O and A498 cells. Data presents as mean ± SD with three replicates. ***, P < 0.001. O , The ChIP was firstly performed by using the YY1 antibodies. Then, the ChIP-re-ChIP assay was performed by using the IgG or HDAC2 antibodies in 786-O and A498 cells. Data presents as mean ± SD with three replicates. ***, P < 0.001. P , 786-O and A498 cells were transfected with the indicated siRNAs for 48 h. Cells were collected for western blot analysis

Article Snippet: Immunohistochemistry was performed with primary antibodies against YTHDC1 (#29441-1-AP, Proteintech, 1:1000, ANXA1 (21990-1-AP, Proteintech; 1:10000 dilution) and TMA slides (#KD1921, Avilabio, China).

Techniques: Expressing, Transfection, Western Blot, Quantitative RT-PCR, ChIP-qPCR, Sequencing, Binding Assay, Mutagenesis, Plasmid Preparation, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway

doi: 10.1186/s13046-022-02460-9

Figure Lengend Snippet: HDAC2 inhibitors enhance the sensitivity of ccRCC to sunitinib. A and B , 786-O and A498 cells were treated with DMSO or CAY10683 (5 µM) for 24 h. Cells were collected for western blot analysis ( A ) and RT-qPCR assay ( B ). Data presents as mean ± SD with three replicates. ***, P < 0.001. C and D , 786-O and A498 cells were transfected with indicated plasmids for 24 h. Then, these cells were treated with DMSO or CAY10683 (5 µM) for another 24 h. Cells were collected for western blot analysis ( C ) and RT-qPCR assay ( D ). Data presents as mean ± SD with three replicates. Ns, not significant; **, P < 0.01; ***, P < 0.001. E and F , 786-O and A498 cells were transfected with indicated siRNAs for 24 h. Then, these cells were treated with DMSO or CAY10683 (5 µM) for another 24 h. Cells were collected for western blot analysis ( C ) and RT-qPCR assay ( D ). Data presents as mean ± SD with three replicates. Ns, not significant; **, P < 0.01; ***, P < 0.001. G , A498 and 786-O cells were transfected with indicated shRNAs for 48 h. Then, these cells were treated with vehicle (DMSO) or CAY10683 (5 µM) for another 24 h. These cells were harvested and treated with a serial dose of sunitinib. These cells were subjected to CCK-8 assay. H , 786-O cells were transfected with indicated shRNAs for 48 h. Then, these cells were treated with vehicle (DMSO) or CAY10683 (5 µM) for another 24 h. These cells were subjected to CCK-8 assay. Data presents as mean ± SD with three replicates. Ns, not significant; **, P < 0.01; ***, P < 0.001. I and J , 786-O cells were transfected with indicated shRNAs for 72 h. After puromycin selection, cells were collected and subcutaneously injected into the nude mice. The tumor mass was shown in panel I, and tumor growth curve was shown in panel J. Data presents as mean ± SD with six replicates. Ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. K , a model depicting that the HDAC2/YY1 complex transcriptionally represses the expression of YTHDC1, which prevents the downregulation of ANXA1 induced by YTHDC1 and activates the MAPK pathway to decrease the sensitivity of ccRCC to sunitinib. HDAC2 inhibitors treatment blocks the HDAC2/YY1/YTHDC1/ANXA1/MAPK axis to enhance the anti-tumor effect of sunitinib

Article Snippet: Immunohistochemistry was performed with primary antibodies against YTHDC1 (#29441-1-AP, Proteintech, 1:1000, ANXA1 (21990-1-AP, Proteintech; 1:10000 dilution) and TMA slides (#KD1921, Avilabio, China).

Techniques: Western Blot, Quantitative RT-PCR, Transfection, CCK-8 Assay, Selection, Injection, Expressing

Journal: Theranostics

Article Title: RNA N 6 -methyladenosine reader YTHDC1 is essential for TGF-beta-mediated metastasis of triple negative breast cancer

doi: 10.7150/thno.71872

Figure Lengend Snippet: YTHDC1 promotes TNBC metastasis. A) Breast cancer patient survival in patients expressing high or low levels of YTHDC1 from TCGA data. Log-rank test. B) Box-whisker plot of YTHDC1 protein levels in tumor and normal patient samples from CPTAC data. Z-values represent standard deviations from the median across samples. t -test. C) Western Blot of YTHDC1 levels across different TNBC cells and non-tumorigenic MCF10A cells. D-F) Cells were engrafted into the 4 th mammary fat pad of 6-9 week-old NSG mice. 1×10 6 MDA-MB-231 cells overexpressing YTHDC1 (OE) or vector were engrafted into each mouse. n = 10/group. D) Quantification of lung metastases by chemiluminescence (top) and associated images (bottom). t -test. E) Number and size of lung metastases (left) and corresponding images of lung samples (right) stained for human CK18. Each point represents the average number or size of 10 metastatic nodules in each mouse. Scale bar: 100 μm. n = 5/group. t -test. F) Volume of primary tumor measured twice weekly. Two-way ANOVA. G-H) 2×10 5 SUM159 cells overexpressing YTHDC1 (OE) or vector were engrafted into each mouse. n = 5/group. G) Quantification of lung metastases by chemiluminescence and associated images. t -test. H) Volume of primary tumor measured twice weekly. Two-way ANOVA. I-J) 1×10 6 MDA-MB-231 cas9 cells transduced with either control sgRNA (sgNS) or sgRNAs targeting YTHDC1 (sgA and sgB) were engrafted into each mouse. n = 5/group. I) Number and size of lung metastases and images of lung samples stained for human CK18. Each point represents the average number or size of 10 metastatic nodules in each mouse. n = 5/group. Scale bar: 100 μm. One-way ANOVA compared to sgNS. J) Volume of primary tumor measured twice weekly. Two-way ANOVA. The data are presented as the mean ± SD for tumor volume plots; NS = not significant, * P < 0.05.

Article Snippet: Primary antibodies used for Western blotting were: YTHDC1 (A305-096A, Bethyl Laboratories), β-Actin (3700S, Cell Signaling Technologies), SMAD2/3 (3102S, Cell Signaling Technologies), SNAI1 (3895S, Cell Signaling Technologies), CDH1 (PA5-80457, Thermo Fisher Scientific), HDAC1 (sc-7872, Santa Cruz Biotechnology), Flag (F3165, Sigma-Aldrich), and GAPDH (10494-1-AP, Proteintech).

Techniques: Expressing, Whisker Assay, Western Blot, Plasmid Preparation, Staining, Transduction, Control

Journal: Theranostics

Article Title: RNA N 6 -methyladenosine reader YTHDC1 is essential for TGF-beta-mediated metastasis of triple negative breast cancer

doi: 10.7150/thno.71872

Figure Lengend Snippet: Identification of YTHDC1 target mRNAs in MDA-MB-231 cells by integrated analysis of sequencing data. A) Gene Ontology (GO) analysis of significantly differentially expressed genes in MDA-MB-231 YTHDC1 KO cells compared to control. Pathways related to metastasis are highlighted in red. B) Pathways related to metastasis enriched in Gene Set Enrichment Analysis (GSEA) of significantly differentially expressed genes in MDA-MB-231 YTHDC1 KO cells compared to control. C) Venn diagram of differentially expressed genes overlapped with RIP-seq and m 6 A-seq data. D) Volcano plot showing changes in mRNA nuclear export following YTHDC1 KO compared to control. E) Venn diagram of nuclear retained transcripts overlapped with RIP-seq and m 6 A-seq data. F) RNA-seq, RIP-seq and m 6 A-seq tracks for SMAD3. G) Correlation between SMAD3 protein and YTHDC1 protein expression levels from CPTAC breast cancer tumor samples. Pearson's correlation test.

Article Snippet: Primary antibodies used for Western blotting were: YTHDC1 (A305-096A, Bethyl Laboratories), β-Actin (3700S, Cell Signaling Technologies), SMAD2/3 (3102S, Cell Signaling Technologies), SNAI1 (3895S, Cell Signaling Technologies), CDH1 (PA5-80457, Thermo Fisher Scientific), HDAC1 (sc-7872, Santa Cruz Biotechnology), Flag (F3165, Sigma-Aldrich), and GAPDH (10494-1-AP, Proteintech).

Techniques: Sequencing, Control, RNA Sequencing, Expressing

Journal: Theranostics

Article Title: RNA N 6 -methyladenosine reader YTHDC1 is essential for TGF-beta-mediated metastasis of triple negative breast cancer

doi: 10.7150/thno.71872

Figure Lengend Snippet: YTHDC1 promotes SMAD3 mRNA nuclear export in TNBC cells. A) SMAD3 transcript levels quantified by RT-qPCR in MDA-MB-231 YTHDC1 KO or SUM159 YTHDC1 KD cells. One-way ANOVA compared to control. B) Changes in the nuclear to cytoplasmic ratio of SMAD3 mRNA in MDA-MB-231 YTHDC1 KO or SUM159 YTHDC1 KD cells quantified by RT-qPCR. One-way ANOVA compared to control. C) Representative images and quantification of SMAD3 mRNA localization by FISH following YTHDC1 KO in MDA-MB-231 cells. Scale bar: 10 μm. Each point on the graph represents a single cell. One-way ANOVA compared to sgNS. n = 50/group. D) Western Blots of SMAD3 in MDA-MB-231 YTHDC1 KO or SUM159 YTHDC1 KD cells. E) m 6 A-RT-qPCR for SMAD3 in MDA-MB-231 and SUM159 cells. HPRT1 was used as a non-target control. One-way ANOVA compared to HPRT1. F) CLIP-RT-qPCR for SMAD3 in MDA-MB-231 and SUM159 cells. HPRT1 was used as a non-target control. One-way ANOVA compared to flag-immunoprecipitated HPRT1. RT-qPCR data are presented as the mean ± SD; n = 3/group, NS = not significant, * P < 0.05.

Article Snippet: Primary antibodies used for Western blotting were: YTHDC1 (A305-096A, Bethyl Laboratories), β-Actin (3700S, Cell Signaling Technologies), SMAD2/3 (3102S, Cell Signaling Technologies), SNAI1 (3895S, Cell Signaling Technologies), CDH1 (PA5-80457, Thermo Fisher Scientific), HDAC1 (sc-7872, Santa Cruz Biotechnology), Flag (F3165, Sigma-Aldrich), and GAPDH (10494-1-AP, Proteintech).

Techniques: Quantitative RT-PCR, Control, Western Blot, Immunoprecipitation

Journal: Theranostics

Article Title: RNA N 6 -methyladenosine reader YTHDC1 is essential for TGF-beta-mediated metastasis of triple negative breast cancer

doi: 10.7150/thno.71872

Figure Lengend Snippet: YTHDC1 is essential for TGF-β-mediated EMT. A-B) Transwell migration and invasion assays for (A) MDA-MB-231 YTHDC1 KO cells (5×10 4 cells/well incubated for 5 hours for migration and 24 hours for invasion) or (B) SUM159 YTHDC1 KD cells (5×10 4 cells/well incubated for 24 hours for migration and invasion) treated with 5 ng/ml TGF-β. One-way ANOVA compared to sgNS (MDA-MB-231) or shNS (SUM159) group. * P < 0.05. C-D) Western Blot for protein quantification from (C) MDA-MB-231 YTHDC1 KO cells or (D) SUM159 YTHDC1 KD cells treated with 5 ng/ml TGF-β for 24 hours. E-F) RT-qPCR of TGF-β responsive genes in (E) MDA-MB-231 YTHDC1 KO cells or (F) SUM159 YTHDC1 KD cells treated with 5 ng/ml TGF-β for 24 hours. One-way ANOVA compared to sgNS (MDA-MB-231) or shNS (SUM159) group. * P < 0.05. G) Western Blot for protein quantification from HCC1806 YTHDC1 KD cells treated with 5 ng/ml TGF-β for 72 hours. H) RT-qPCR of TGF-β responsive genes in HCC1806 YTHDC1 KD cells treated with 5 ng/ml TGF-β for 72 hours. One-way ANOVA compared to shNS group. NS = not significant, * P < 0.05. I) Images and quantification of the shapes of control, YTHDC1 MDA-MB-231 KO and SUM159 KD cells treated with TGF-β or vehicle control for 4 days and stained with vimentin. Scale bar: 50 μm. A larger aspect ratio represents a more elongated cell shape. One-way ANOVA compared to control shNS group. NS = not significant, * P < 0.05.

Article Snippet: Primary antibodies used for Western blotting were: YTHDC1 (A305-096A, Bethyl Laboratories), β-Actin (3700S, Cell Signaling Technologies), SMAD2/3 (3102S, Cell Signaling Technologies), SNAI1 (3895S, Cell Signaling Technologies), CDH1 (PA5-80457, Thermo Fisher Scientific), HDAC1 (sc-7872, Santa Cruz Biotechnology), Flag (F3165, Sigma-Aldrich), and GAPDH (10494-1-AP, Proteintech).

Techniques: Migration, Incubation, Western Blot, Quantitative RT-PCR, Control, Staining

Journal: Theranostics

Article Title: RNA N 6 -methyladenosine reader YTHDC1 is essential for TGF-beta-mediated metastasis of triple negative breast cancer

doi: 10.7150/thno.71872

Figure Lengend Snippet: YTHDC1 promotes TGF-β-mediated metastasis by augmenting SMAD3 levels. (A) MDA-MB-231 cas9 cells were engrafted into the 4 th mammary fat pad of 6-9 week-old NSG mice. 2×10 6 cells expressing either vector + sgNS, vector + sgA, or SMAD3 overexpression + sgA were engrafted into each mouse. n = 5/group. Associated lung images (top) and quantification of chemiluminescence signal (bottom left). One-way ANOVA compared to vector + sgNS group. NS = not significant, * P < 0.05. Tumor volumes are also shown (bottom right). Two-way ANOVA compared to vector + sgNS group. NS = not significant, * P < 0.05. B-C) Transwell migration and invasion assays for (B) MDA-MB-231 YTHDC1 KO cells (4×10 4 cells/well incubated for 24 hours for migration and invasion) or (C) SUM159 YTHDC1 KD (4×10 4 cells/well incubated for 24 hours for migration and invasion) cells overexpressing SMAD3 treated with 5 ng/ml TGF-β. One-way ANOVA compared to vector + sgNS/shNS group. NS = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. D-E) Western Blot for protein quantification from (D) MDA-MB-231 YTHDC1 KO cells or (E) SUM159 YTHDC1 KD cells overexpressing SMAD3 treated with 5 ng/ml TGF-β for 24 hours. F-G) RT-qPCR of TGF-β responsive genes in (F) MDA-MB-231 YTHDC1 KO cells or (G) SUM159 YTHDC1 KD cells overexpressing SMAD3 treated with 5 ng/ml TGF-β for 24 hours. One-way ANOVA compared to vector + sgNS/shNS group. * P < 0.05. The data are presented as the mean ± SD; n = 3/group.

Article Snippet: Primary antibodies used for Western blotting were: YTHDC1 (A305-096A, Bethyl Laboratories), β-Actin (3700S, Cell Signaling Technologies), SMAD2/3 (3102S, Cell Signaling Technologies), SNAI1 (3895S, Cell Signaling Technologies), CDH1 (PA5-80457, Thermo Fisher Scientific), HDAC1 (sc-7872, Santa Cruz Biotechnology), Flag (F3165, Sigma-Aldrich), and GAPDH (10494-1-AP, Proteintech).

Techniques: Expressing, Plasmid Preparation, Over Expression, Migration, Incubation, Western Blot, Quantitative RT-PCR

Journal: Theranostics

Article Title: RNA N 6 -methyladenosine reader YTHDC1 is essential for TGF-beta-mediated metastasis of triple negative breast cancer

doi: 10.7150/thno.71872

Figure Lengend Snippet: A functional m 6 A-binding domain is necessary for the tumorigenic functions of YTHDC1. A) Western Blot analysis of YTHDC1 and SMAD3 in MDA-MB-231 control or YTHDC1 KO cells overexpressing either empty vector, wild type (WT), or m 6 A-binding defective mutants (W377A and W428A) of YTHDC1. B) Transwell migration and invasion assays of MDA-MB-231 control (sgNS) or YTHDC1 KO cells overexpressing either empty vector, wild type (WT), or mutant (W377A or W428A) YTHDC1 (5×10 4 cells/well incubated for 24 hours for migration and invasion). One-way ANOVA compared to Vector/sgNS group. C) CLIP-RT-qPCR of SMAD3 bound to wild type (WT) or mutant (W377A or W428A) YTHDC1 in MDA-MB-231 cells. One-way ANOVA compared to WT group. D) Western Blot for immunoprecipitation of wild type or mutant YTHDC1 using anti-flag or control IgG antibodies in CLIP-RT-qPCR assay. (E) Dual luciferase assay in HEK293T cells transfected with WT or mutant SMAD3 3'UTR fused to a firefly luciferase (Fluc) gene. Renilla luciferase (Rluc) used as a loading control. One-way ANOVA compared to shNS+WT SMAD3 3'UTR. (F) Changes in the nuclear to cytoplasmic ratio of firefly luciferase mRNA in HEK293T cells quantified by RT-qPCR. One-way ANOVA compared to shNS+WT SMAD3 3'UTR. The data are presented as the mean ± SD; n = 3/group. NS = not significant, * P < 0.05. (G) Western Blot assay showing nuclear and cytoplasmic distribution of YTHDC1 protein in HEK293T cells and YTHDC1 KD efficiency. HDAC1 and GAPDH were used as nuclear and cytoplasmic marker, respectively.

Article Snippet: Primary antibodies used for Western blotting were: YTHDC1 (A305-096A, Bethyl Laboratories), β-Actin (3700S, Cell Signaling Technologies), SMAD2/3 (3102S, Cell Signaling Technologies), SNAI1 (3895S, Cell Signaling Technologies), CDH1 (PA5-80457, Thermo Fisher Scientific), HDAC1 (sc-7872, Santa Cruz Biotechnology), Flag (F3165, Sigma-Aldrich), and GAPDH (10494-1-AP, Proteintech).

Techniques: Functional Assay, Binding Assay, Western Blot, Control, Plasmid Preparation, Migration, Mutagenesis, Incubation, Quantitative RT-PCR, Immunoprecipitation, Luciferase, Transfection, Marker

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Comprehensive immunohistochemical analysis of N6-methyladenosine (m6A) writers, erasers, and readers in endometrial cancer

doi: 10.1007/s00432-022-04083-1

Figure Lengend Snippet: Summary of analyzed proteins and their associations with overall survival

Article Snippet: Immunostaining of METTL3, METTL4, METTL14, WTAP, KIAA1429, FTO, ALKBH5, HNRNPA2B1, HNRNPC, YTHDC1, YTHDF1,YTHDF2, and YTHDF3 was performed on 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: