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: Molecular cell

Article Title: N6 -methyladenosine modification controls circular RNA immunity

doi: 10.1016/j.molcel.2019.07.016

Figure Lengend Snippet: A. YTHDF2 is necessary for differential response to unmodified vs. m6A-modified circFOREIGN. Transfection of unmodified or m6A-modified circFOREIGN into YTHDF2−/− HeLa cells stimulate immune response. Left: Model showing the responses to unmodified or m6A-modified circFOREIGN. Right: Graph showing gene expression of innate immune genes 24 hours following RNA transfection. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR, results normalized to expression following mock transfection. Means ± SEM are shown (n = 3). Student’s t-test, comparing circFOREIGN with 0% m6A to indicated RNA transfection. B. Ectopic expression of YTHDF2 rescues response to unmodified vs. m6A-modified circFOREIGN in YTHDF2 KO HeLa cells. Left: Model showing response to m6A-modified circFOREIGN following rescue. Right: Graph showing gene expression of innate immune genes 24 hours following RNA transfection. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR, results normalized to expression following mock transfection. Means ± SEM are shown (n = 3). *p<0.05, Student’s t-test, comparing 0% m6A circFOREIGN to 1% m6A circFOREIGN. C. Tethering of YTHDF2 to unmodified circFOREIGN mask circRNA immunity. Left: Model showing in vivo tethering of protein to RNA via lambdaN and BoxB leading to attenuation of immunogencity. Right top: Protein domain architecture of full-length wild-type YTHDF2 with and without lambdaN tethering tag, and YTHDF2 N-terminal domain with and without lambdaN tethering tag. Right bottom: RIP-qPCR enrichment of indicated YTH protein followed by qRT-PCR of circRNA-BoxB or control actin RNA. Means ± SEM are shown (n = 3). *p<0.05, Student’s t-test, comparing YTHDF2 N-terminus with lambdaN tethering to YTHDF2 N-terminus without tethering. D. Transfection of unmodified circBoxB tethered to full length wild-type YTHDF2 into wild-type HeLa cells attenuates immune response. Graph showing gene expression of innate immune genes 24 hours following RNA transfection. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR, results normalized mock transfection. Wild-type YTHDF2-lambdaN (grey) was ectopically expressed as immunogenicity negative control. Transfection with solely circBoxB (purple) serves as immunogenicity positive control. Means ± SEM are shown (n = 3). *p<0.05, Student’s t-test, comparing circBoxB with wild-type YTHDF2 with lambdaN tethering to wild-type YTHDF2 without tethering. E. Transfection of unmodified circBoxB tethered to N-terminal domain of YTHDF2 into YTHDF2 KO cells is insufficient to attenuate immune response. Graph showing gene expression of innate immune genes 24 hours following RNA transfection. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR, results normalized mock transfection. N-terminal domain of YTHDF2-lambdaN (black) was ectopically expressed as immunogenicity negative control. Means ± SEM are shown (n = 3). Student’s t-test, comparing circBoxB with YTHDF2 N-terminus with lambdaN tethering to YTHDF2 N-terminus without tethering.

Article Snippet: Mouse Anti-YTHDF2 polyclonal antibody (1:200 for IF) , USBiological Life Sciences , 135486 RRID: N/A.

Techniques: Modification, Transfection, Gene Expression, Expressing, Quantitative RT-PCR, In Vivo, Control, Immunopeptidomics, Negative Control, Positive Control

Journal: Molecular cell

Article Title: N6 -methyladenosine modification controls circular RNA immunity

doi: 10.1016/j.molcel.2019.07.016

Figure Lengend Snippet: A. CircFOREIGN co-localizes with RIG-I and K63-polyubiquitin chain. Representative field of view is shown. B. Quantification of circFOREIGN colocalization with RIG-I and K63-Ubn (n = 152). Foci were collected across 10 fields of view across biological replicates and representative of replicate experiments. C. 10% m6A circFOREIGN has increased co-localization with YTHDF2. Representative field of view is shown. Foci were collected across >10 fields of view and representative of replicate experiments. D. Quantification of circFOREIGN and 10% m6A circFOREIGN colocalization with YTHDF2 and RIG-I. *p<0.05, Pearson’s χ² test.

Article Snippet: Mouse Anti-YTHDF2 polyclonal antibody (1:200 for IF) , USBiological Life Sciences , 135486 RRID: N/A.

Techniques:

Journal: Molecular cell

Article Title: N6 -methyladenosine modification controls circular RNA immunity

doi: 10.1016/j.molcel.2019.07.016

Figure Lengend Snippet: Unmodified circRNA interacts with RIG-I in the presence of K63-Ubn to induce MAVS filamentation, which triggers IRF3 dimerization and interferon production. m6A-modified circRNAs together with YTHDF2 repel RIG-I and does not initiate signaling.

Article Snippet: Mouse Anti-YTHDF2 polyclonal antibody (1:200 for IF) , USBiological Life Sciences , 135486 RRID: N/A.

Techniques: Modification

Journal: Molecular cell

Article Title: N6 -methyladenosine modification controls circular RNA immunity

doi: 10.1016/j.molcel.2019.07.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse Anti-YTHDF2 polyclonal antibody (1:200 for IF) , USBiological Life Sciences , 135486 RRID: N/A.

Techniques: Produced, Recombinant, Purification, Magnetic Beads, Virus, Cloning, Protease Inhibitor, Immunoprecipitation, Binding Assay, Sterility, Enzyme-linked Immunospot, Transfection, Membrane, Plasmid Preparation, Saline, Staining, Sequencing, Control, Software, Microscopy