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