anti-ythdf2 polyclonal antibody fnab09573  (FineTest Biotech Inc)

Journal: International Journal of Molecular Sciences

Article Title: BMP9-ID1 Pathway Attenuates N 6 -Methyladenosine Levels of CyclinD1 to Promote Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/ijms25020981

Figure Lengend Snippet: BMP9 enhances CyclinD1 expression in HCC cells to facilitate cell cycle progression via suppressing m 6 A methylation within the 5′ UTR of CyclinD1 mRNA ( A ) CCK-8 assay was used to evaluate the cell proliferation of Huh7 and Hep3B cells. ( B ) Colony formation assay was used to evaluate the cell proliferation of Huh7 and Hep3B cells. Scale bar = 1 cm ( C ) Flow cytometry was used to analyze cell cycle of Huh7 and Hep3B cells. ( D ) m 6 A dot blotting was used to evaluate the global RNA m 6 A methylation of Huh7 and Hep3B cells. ( E ) Relative gene expression levels of ID1, CyclinD1, FTO and YTHDF2 in Huh7 and Hep3B cells. ( F ) Western blot analysis of ID1, CyclinD1, FTO and YTHDF2 in Huh7 and Hep3B cells. ( G ) The m 6 A methylation within the 5′-UTR of CyclinD1 mRNA in Huh7 and Hep3B cells was analyzed using MeRIP-qPCR. Cells underwent treatment with either Dimethyl Sulfoxide (DMSO) or BMP9 (5 ng/mL) for a duration of 48 h. The error bars illustrate the Standard Deviation (SD) derived from a minimum of three independent biological repetitions. Student’s t -test was employed to compute the p -values, depicted as * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: For Western blotting, the following primary antibodies were utilized: anti-ID1 monoclonal antibody (sc-133104, Santa Cruz, Dallas, TX, USA), anti-CyclinD1 monoclonal antibody (92G2, Cell Signaling Technology), anti-FTO monoclonal antibody (ab126605, Abcam, Cambridge, UK), anti-YTHDF2 polyclonal antibody (FNab09573, FineTest, Guangzhou, China), and anti-β-actin monoclonal antibody (Zsbio, Beijing, China).

Techniques: Expressing, Methylation, CCK-8 Assay, Colony Assay, Flow Cytometry, Western Blot, Standard Deviation, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: BMP9-ID1 Pathway Attenuates N 6 -Methyladenosine Levels of CyclinD1 to Promote Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/ijms25020981

Figure Lengend Snippet: ID1 enhances cell cycle progression and inhibits m 6 A methylation within the 5′ UTR of CyclinD1 mRNA. ( A ) CCK-8 assay was used to evaluate the cell proliferation of Huh7 and Hep3B cells. ( B ) Colony formation assay was used to evaluate the cell proliferation of Huh7 and Hep3B cells. Scale Bar = 1 cm ( C ) Flow cytometry was used to analyze cell cycle of Huh7 and Hep3B cells. ( D ) m 6 A dot blotting was used to evaluate the global RNA m 6 A methylation of Huh7 and Hep3B cells. ( E ) Relative gene expression levels of ID1, CyclinD1, FTO and YTHDF2 in Huh7 and Hep3B cells. ( F ) Western blot analysis of ID1, CyclinD1, FTO and YTHDF2 in Huh7 and Hep3B cells. ( G ) The m 6 A methylation within the 5′-UTR of CyclinD1 mRNA in Huh7 and Hep3B cells was analyzed using MeRIP-qPCR. Cells were transfected 2 μg ID1 plasmid or vector for 48 h. The error bars denote the Standard Deviation (SD) drawn from a minimum of three separate biological replicates. The p -values were computed using Student’s t -test, represented as ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: For Western blotting, the following primary antibodies were utilized: anti-ID1 monoclonal antibody (sc-133104, Santa Cruz, Dallas, TX, USA), anti-CyclinD1 monoclonal antibody (92G2, Cell Signaling Technology), anti-FTO monoclonal antibody (ab126605, Abcam, Cambridge, UK), anti-YTHDF2 polyclonal antibody (FNab09573, FineTest, Guangzhou, China), and anti-β-actin monoclonal antibody (Zsbio, Beijing, China).

Techniques: Methylation, CCK-8 Assay, Colony Assay, Flow Cytometry, Expressing, Western Blot, Transfection, Plasmid Preparation, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: BMP9-ID1 Pathway Attenuates N 6 -Methyladenosine Levels of CyclinD1 to Promote Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/ijms25020981

Figure Lengend Snippet: Knockdown of ID1 suppresses cell cycle progression and induces m 6 A methylation within the 5′ UTR of CyclinD1 mRNA. ( A ) CCK-8 assay was used to evaluate the cell proliferation of Huh7 and Hep3B cells. ( B ) Colony formation assay was used to evaluate the cell proliferation of Huh7 and Hep3B cells. Scale bar = 1 cm ( C ) Flow cytometry was used to analyze cell cycle of Huh7 and Hep3B cells. ( D ) m 6 A dot blotting was used to evaluate the global RNA m 6 A methylation of Huh7 and Hep3B cells. ( E ) Relative gene expression levels of ID1, CyclinD1, FTO and YTHDF2 in Huh7 and Hep3B cells. ( F ) Western blot analysis of ID1, CyclinD1, FTO and YTHDF2 in Huh7 and Hep3B cells. ( G ) The m 6 A methylation within the 5′ UTR of CyclinD1 mRNA in Huh7 and Hep3B cells was analyzed using MeRIP-qPCR. Cells were transfected 20 nM ID1 siRNA or siCtrl for 48 h. The error bars denote the Standard Deviation (SD) drawn from a minimum of three separate biological replicates. The p -values were computed using Student’s t -test, represented as * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: For Western blotting, the following primary antibodies were utilized: anti-ID1 monoclonal antibody (sc-133104, Santa Cruz, Dallas, TX, USA), anti-CyclinD1 monoclonal antibody (92G2, Cell Signaling Technology), anti-FTO monoclonal antibody (ab126605, Abcam, Cambridge, UK), anti-YTHDF2 polyclonal antibody (FNab09573, FineTest, Guangzhou, China), and anti-β-actin monoclonal antibody (Zsbio, Beijing, China).

Techniques: Methylation, CCK-8 Assay, Colony Assay, Flow Cytometry, Expressing, Western Blot, Transfection, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: BMP9-ID1 Pathway Attenuates N 6 -Methyladenosine Levels of CyclinD1 to Promote Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/ijms25020981

Figure Lengend Snippet: Knockdown of ID1 attenuates the upregulated progression of cell cycle and the downregulated m 6 A methylation within the 5′ UTR of CyclinD1 mRNA induced by BMP9 in HCC cells. ( A ) CCK-8 assay was used to evaluate the cell proliferation of Huh7 and Hep3B cells. ( B ) Colony formation assay was used to evaluate the cell proliferation of Huh7 and Hep3B cells. Scale bar = 1 cm ( C ) Flow cytometry was used to analyze cell cycle of Huh7 and Hep3B cells. ( D ) m 6 A dot blotting was used to evaluate the global RNA m 6 A methylation of Huh7 and Hep3B cells. ( E ) Relative gene expression levels of ID1, CyclinD1, FTO and YTHDF2 in Huh7 and Hep3B cells. ( F ) Western blot analysis of ID1, CyclinD1, FTO and YTHDF2 in Huh7 and Hep3B cells. ( G ) The m 6 A methylation within the 5′ UTR of CyclinD1 mRNA in Huh7 and Hep3B cells was analyzed using MeRIP-qPCR. Cells were treated with BMP9 treated with or without BMP9 (5 ng/mL) for 48 h following siID1 or siCtrl transfection. The error bars denote the Standard Deviation (SD) drawn from a minimum of three separate biological replicates. The p -values were computed using Student’s t -test, represented as * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: For Western blotting, the following primary antibodies were utilized: anti-ID1 monoclonal antibody (sc-133104, Santa Cruz, Dallas, TX, USA), anti-CyclinD1 monoclonal antibody (92G2, Cell Signaling Technology), anti-FTO monoclonal antibody (ab126605, Abcam, Cambridge, UK), anti-YTHDF2 polyclonal antibody (FNab09573, FineTest, Guangzhou, China), and anti-β-actin monoclonal antibody (Zsbio, Beijing, China).

Techniques: Methylation, CCK-8 Assay, Colony Assay, Flow Cytometry, Expressing, Western Blot, Transfection, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: BMP9-ID1 Pathway Attenuates N 6 -Methyladenosine Levels of CyclinD1 to Promote Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/ijms25020981

Figure Lengend Snippet: BMP receptor inhibitors repress cell cycle progression and promote m 6 A methylation within the 5′ UTR of CyclinD1 mRNA in HCC cells. ( A ) CCK-8 assay was used to evaluate the cell proliferation of Huh7 and Hep3B cells. ( B ) Colony formation assay was used to evaluate the cell proliferation of Huh7 and Hep3B cells. Scale bar = 1 cm ( C ) Flow cytometry was used to analyze cell cycle of Huh7 and Hep3B cells. ( D ) m 6 A dot blotting was used to evaluate the global RNA m 6 A methylation of Huh7 and Hep3B cells. ( E ) Relative gene expression levels of ID1, CyclinD1, FTO and YTHDF2 in Huh7 and Hep3B cells. ( F ) Western blot analysis of ID1, CyclinD1, FTO and YTHDF2 in Huh7 and Hep3B cells. ( G ) The m 6 A methylation within the 5′-UTR of CyclinD1 mRNA in Huh7 and Hep3B cells was analyzed using MeRIP-qPCR. Cells were treated with 2 μM K02288, LDN-212854 or DMSO for 48 h. The error bars denote the Standard Deviation (SD) drawn from a minimum of three separate biological replicates. The p -values were computed using Student’s t -test, represented as * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: For Western blotting, the following primary antibodies were utilized: anti-ID1 monoclonal antibody (sc-133104, Santa Cruz, Dallas, TX, USA), anti-CyclinD1 monoclonal antibody (92G2, Cell Signaling Technology), anti-FTO monoclonal antibody (ab126605, Abcam, Cambridge, UK), anti-YTHDF2 polyclonal antibody (FNab09573, FineTest, Guangzhou, China), and anti-β-actin monoclonal antibody (Zsbio, Beijing, China).

Techniques: Methylation, CCK-8 Assay, Colony Assay, Flow Cytometry, Expressing, Western Blot, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: BMP9-ID1 Pathway Attenuates N 6 -Methyladenosine Levels of CyclinD1 to Promote Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/ijms25020981

Figure Lengend Snippet: BMP receptor inhibitors attenuate upregulated cell cycle progression and downregulated m 6 A methylation within 5′ UTR of CyclinD1 mRNA induced by BMP9 in HCC cells. ( A ) CCK-8 assay was used to evaluate the cell proliferation of Huh7 and Hep3B cells. Scale bar = 1 cm ( B ) Colony formation assay was used to evaluate the cell proliferation of Huh7 and Hep3B cells. ( C ) Flow cytometry was used to analyze cell cycle of Huh7 and Hep3B cells. ( D ) m 6 A dot blotting was used to evaluate the global RNA m 6 A methylation of Huh7 and Hep3B cells. ( E ) Relative gene expression levels of ID1, CyclinD1, FTO and YTHDF2 in Huh7 and Hep3B cells. ( F ) Western blot analysis of ID1, CyclinD1, FTO and YTHDF2 in Huh7 and Hep3B cells. ( G ) The m 6 A methylation within the 5′ UTR of CyclinD1 mRNA in Huh7 and Hep3B cells was analyzed using MeRIP-qPCR. Cells were treated with 2 μM K02288, LDN-212854 or DMSO in the presence of BMP9 (5 ng/mL) for 48 h. The error bars denote the Standard Deviation (SD) drawn from a minimum of three separate biological replicates. The p -values were computed using Student’s t -test, represented as * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: For Western blotting, the following primary antibodies were utilized: anti-ID1 monoclonal antibody (sc-133104, Santa Cruz, Dallas, TX, USA), anti-CyclinD1 monoclonal antibody (92G2, Cell Signaling Technology), anti-FTO monoclonal antibody (ab126605, Abcam, Cambridge, UK), anti-YTHDF2 polyclonal antibody (FNab09573, FineTest, Guangzhou, China), and anti-β-actin monoclonal antibody (Zsbio, Beijing, China).

Techniques: Methylation, CCK-8 Assay, Colony Assay, Flow Cytometry, Expressing, Western Blot, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: BMP9-ID1 Pathway Attenuates N 6 -Methyladenosine Levels of CyclinD1 to Promote Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/ijms25020981

Figure Lengend Snippet: BMP receptor inhibitor LDN-212854 represses tumor growth and promotes global RNA m 6 A methylation of HCC xenografts. ( A ) Influence of LDN-212854 on growth of Huh7 xenograft tumors. Mice bearing Huh7 xenograft tumors were treated with PBS (n = 5) or LDN-212854 (n = 5). ( B ) Influence of LDN-212854 on growth of Hep3B xenograft tumors. Mice bearing Hep3B xenograft tumors were treated with PBS (n = 6) or LDN-212854 (n = 6). ( C ) m 6 A dot blotting showed the global RNA m 6 A methylation in Huh7 and Hep3B xenograft tumors. ( D , E ) Western blot analysis of CyclinD1, ID1, FTO and YTHDF2 expressions in Huh7 and Hep3B xenograft tumors. β-actin was used as the reference for quantifying protein expression. ( F ) IHC analysis of ID1, CyclinD1, FTO and YTHDF2 expression in Huh7 and Hep3B xenograft tumors. Scale bar = 200 μm. The error bars denote the Standard Deviation (SD) drawn from a minimum of three separate biological replicates. The p -values were computed using Student’s t -test, represented as * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: For Western blotting, the following primary antibodies were utilized: anti-ID1 monoclonal antibody (sc-133104, Santa Cruz, Dallas, TX, USA), anti-CyclinD1 monoclonal antibody (92G2, Cell Signaling Technology), anti-FTO monoclonal antibody (ab126605, Abcam, Cambridge, UK), anti-YTHDF2 polyclonal antibody (FNab09573, FineTest, Guangzhou, China), and anti-β-actin monoclonal antibody (Zsbio, Beijing, China).

Techniques: Methylation, Western Blot, Expressing, Standard Deviation

Journal: Journal of Clinical Investigation

Article Title: Epitranscriptional regulation of TGF-β pseudoreceptor BAMBI by m6A/YTHDF2 drives extrinsic radioresistance

doi: 10.1172/jci172919

Figure Lengend Snippet: Figure 2. IR/YTHDF2 axis regulates BAMBI expression in MDSCs in an m6A-dependent manner. (A) Integrative Genomics Viewer tracks displaying the distribution of m6A peaks and YTHDF2-binding peaks across Bambi transcripts, based on the MeRIP-Seq and RIP-Seq of MC38 tumor–infiltrating myeloid cells. (B) Graphs showing enrichment of Bambi mRNA in the YTHDF2-immunoprecipitated RNA fraction of BM-MDSCs, determined by RIP-qPCR. Rabbit IgG served as a control. Enrichment of the indicated genes was normalized to the input level. n = 3 per group. (C) MDSCs were sorted from BM-derived cells from WT and Ythdf2-cKO (Lyz2creYthdf2fl/fl) mice; WT MDSCs were directly treated with IR (4 Gy and cultured for 6 hours) (WT+IR). MDSCs were treated with actinomycin D. mRNA was collected at indicated time points after treatment and mRNA levels of Bambi were measured by RT-qPCR. n = 3 per group. (D) qPCR analysis of Bambi mRNA levels in different MDSCs isolated from MC38 tumors in WT, WT+IR, Ythdf2-cKO, and Ythdf2-cKO+IR mice 3 days after IR. n = 5 per group. (E) MFI of BAMBI in MC38 tumor–infiltrating MDSCs (as indicated) by flow cytometry analysis 3 days after IR. n = 6 per group. (F) WT-YTHDF2 and m6A-binding site-mutated YTHDF2-overexpressing Ythdf2-deficient CD45.2-BM-MDSCs (Ythdf2-cKO+WT, Ythdf2-cKO+Mut, respectively) were obtained via lentiviral transfection. The BM-MDSCs were used for adoptive transfer into MC38 tumor–bearing CD45.1 mice. On the same day, mice were treated with local tumor irradiation. Three days after IR, tumors were harvested to measure the MFI of BAMBI in newly infiltrated CD45.2-MDSCs by flow cytometry. n = 5 per group. Data are represented as means ± SEM. One of 2 or 3 representative experiments is shown (B–F). Statistical analysis was performed using 2-sided, unpaired Student’s t test (B) or 1-way ANOVA with Bonferroni’s multiple-comparison tests (C–F). *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: RIP for YTHDF2 was performed using 10 to 20 μg anti-YTHDF2 rabbit polyclonal antibody (catalog ARP67917_P050; Aviva Systems Biology).

Techniques: Expressing, Binding Assay, Immunoprecipitation, Control, Derivative Assay, Cell Culture, Quantitative RT-PCR, Isolation, Flow Cytometry, Transfection, Adoptive Transfer Assay, Irradiation, Comparison