anti ythdc1 primary antibody  (Proteintech)

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 blocked with FBS and incubated overnight with an anti-YTHDC1 primary antibody (#29441-1-AP, Proteintech) at a 1:500 dilution.

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 blocked with FBS and incubated overnight with an anti-YTHDC1 primary antibody (#29441-1-AP, Proteintech) at a 1:500 dilution.

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 blocked with FBS and incubated overnight with an anti-YTHDC1 primary antibody (#29441-1-AP, Proteintech) at a 1:500 dilution.

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 blocked with FBS and incubated overnight with an anti-YTHDC1 primary antibody (#29441-1-AP, Proteintech) at a 1:500 dilution.

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 blocked with FBS and incubated overnight with an anti-YTHDC1 primary antibody (#29441-1-AP, Proteintech) at a 1:500 dilution.

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 blocked with FBS and incubated overnight with an anti-YTHDC1 primary antibody (#29441-1-AP, Proteintech) at a 1:500 dilution.

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