Journal: bioRxiv

Article Title: Regulation and Function of the HPV16 CircE7 RNA

doi: 10.64898/2026.03.09.710444

Figure Lengend Snippet: (A) Western blot from MDA-1483 cells transfected with WT or IRES mut circE7 plasmid. Representative blot (left), quantification (right). ***p<0.001 (B) RNA levels of the IRES mutant cricE7 RNA by RT-qPCR in MDA-1483 cells. Difference not statistically significant by t-test. Twice as much RNA was transfected in to get equivalent levels of RNA between IRES and WT. (C) Representative tracing of circE7-transfected cells after polysome enrichment assay with the monosome (M), light polysome (L), and heavy polysome (H) fractions indicated. Polysomes isolated from SCC-154 HPV+ (red) and MDA1483 HPV- (blue) cells. (D) Endpoint PCR of CircE7 or circHPK3, in light and heavy polysomes from SCC-154 (HPV+) cells, MDA1483 (HPV-) cells, and BJ cells transfected with hTERT circE7. All samples were RNAse R treated. (E) Western blot from HeLa cells co-transfected with a circE7-Flag expression vector and the indicated siRNA. HSP90, loading control. *indicates non-specific band. (F) RT-qPCR for circE7 RNA levels after YTHDC1 knockdown. (G) Quantification of western blot (n=3).

Article Snippet: Membranes were incubated overnight at 4°C with the following primary antibodies: YTHDC1 (Cell Signaling, 77422), YTHDF3 (Cell Signaling, 24206), YTHDC2 (Cell Signaling, 46324), FLAG (Sigma, A8592), GAPDH (Santa Cruz, sc-32233), HSP90 (Cell Signaling, 4877S).

Techniques: Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, Isolation, Expressing, Control, Knockdown

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: Protein-Protein interactions, RNA Sequencing, 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, 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, ChIP-qPCR, 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: 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