Journal: eLife

Article Title: Metabolic clogging of mannose triggers dNTP loss and genomic instability in human cancer cells

doi: 10.7554/eLife.83870

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-MCM5 (rabbit polyclonal) , GeneTex , GTX33310 , WB (1:5000).

Techniques: Retroviral, Recombinant, Drug discovery, Plasmid Preparation, Expressing, Sequencing, CRISPR, Lactate Dehydrogenase Assay, Cloning, Protease Inhibitor, Software

Journal: Advanced Science

Article Title: m 6 A‐Dependent Modulation via IGF2BP3/MCM5/Notch Axis Promotes Partial EMT and LUAD Metastasis

doi: 10.1002/advs.202206744

Figure Lengend Snippet: MCM5 contributed to IGF2BP3 activated Notch signaling in m 6 A dependent manner. A) Overlapping the RNA‐seq dataset of METTL3‐silenced A549 cells with two distinct MeRIP‐seq datasets (GSE29714 and GSE54365) reveals 1137 transcripts (left) and overlapping IGF2BP3‐RIP‐seq (GSE90639) and shIGF2BP3‐RNA‐seq (GSE90684) datasets reveals 356 transcripts (middle). 51 genes are IGF2BP3‐recognized and m 6 A‐modified transcripts, among which only MCM5 emerges as a potential NICD1‐interactive partner (right). B) Co‐IP assay shows interaction between MCM5 and NICD1 proteins. C) Pull‐down assay demonstrates a direct binding between recombinant MCM5 and NICD1. D) Representative images show the interaction and subcellular co‐localization of MCM5 and NICD1 proteins in LUAD cells. Scale bar: 5 µm. E–J) Luciferase reporter and WB assays examining Notch activity and NICD1 levels. All experiments were repeated three times with similar results. Statistical analyses were performed using a two‐tailed Student's t ‐test, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Article Snippet: The primary antibodies used for PLA included MCM5 (Proteintech, Chicago, IL, USA) and cleaved‐Notch1 (Immunoway, plano, TX, USA).

Techniques: RNA Sequencing, Modification, Co-Immunoprecipitation Assay, Pull Down Assay, Binding Assay, Recombinant, Luciferase, Activity Assay, Two Tailed Test

Journal: Advanced Science

Article Title: m 6 A‐Dependent Modulation via IGF2BP3/MCM5/Notch Axis Promotes Partial EMT and LUAD Metastasis

doi: 10.1002/advs.202206744

Figure Lengend Snippet: IGF2BP3 reads m6A‐modified MCM5 mRNA to promote its stability. A) Analysis of m 6 A modification peak based on IGF2BP3‐RIP‐seq (GSE90639) and MeRIP‐seq (GSE54365) datasets. B) MeRIP‐qPCR assay demonstrates enrichment of m 6 A modification in MCM5 3′UTR in A549 cells silenced with control (NC) or METTL3. C) RIP‐qPCR detecting the binding of IGF2BP3 with MCM5 mRNA 3′UTR in A549 and HEK293FT cells. D) The threshold cycle (Ct) of qPCR shows SELECT results for detecting two m 6 A sites in 3′UTR of MCM5 in A549 cells with or without METTL3 knockdown. E,F) MCM5 levels determined by qRT‐PCR and WB assays. G) qRT‐PCR assay showing MCM5 mRNA stability. H) The effect of silencing HuR on MCM5 expression. I) Schematic diagram of wild‐type (WT) or mutant (MUT) MCM5 3′UTR fused to firefly luciferase reporter. J) Luciferase activities affected by wild‐type or mutant MCM5 3′UTR. K) RIP‐qPCR detecting binding of IGF2BP3 to the MCM5 3′UTR. L) qRT‐PCR assay showing MCM5 mRNA stability. M,N) qRT‐PCR assays showing effects of dCas13b‐ALKBH5 on MCM5 mRNA level and stability. O) Luciferase reporter assay detecting Notch signaling activities. All experiments were repeated three times with similar results. Statistical analyses were performed using a two‐tailed Student's t ‐test, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Article Snippet: The primary antibodies used for PLA included MCM5 (Proteintech, Chicago, IL, USA) and cleaved‐Notch1 (Immunoway, plano, TX, USA).

Techniques: Modification, Control, Binding Assay, Knockdown, Quantitative RT-PCR, Expressing, Mutagenesis, Luciferase, Reporter Assay, Two Tailed Test

Journal: Advanced Science

Article Title: m 6 A‐Dependent Modulation via IGF2BP3/MCM5/Notch Axis Promotes Partial EMT and LUAD Metastasis

doi: 10.1002/advs.202206744

Figure Lengend Snippet: m6A modified MCM5 is crucial for IGF2BP3‐induced cancer cell plasticity. A) H&E staining shows muscle penetration in subcutaneous tumor xenografts formed by indicated cells (1 × 10 6 ). n = 5. B–D) Bioluminescent images, picric acid staining and H&E staining show lung metastases formed by tail vein injection with indicated cells. n = 5. E) qRT‐PCR analysis examining p‐EMT and lineage plasticity markers. F–I) Indicated cells (1×10 6 ) were injected subcutaneously (F) or intravenously (G–I), and representative bioluminescent images, picric acid staining and H&E staining of lung metastasis are shown. n = 5. J–M) Indicated cells (1 × 10 6 ) were injected subcutaneously (J) or intravenously (K–M), and representative bioluminescent images, picric acid staining and H&E staining of lung metastases are shown. n = 5. Scale bars, 50 µm. N) Transwell assay showing the effect of inhibiting Notch on MCM5‐augmented invasion. For A, F, and J, the number of mice with local invasion was counted. For B–D, G, H, and K–M, the bioluminescent intensities and the number of lung surface nodules and metastatic lesions in mouse lung tissue were provided. Statistical analyses were performed using a two‐tailed Student's t ‐test, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Article Snippet: The primary antibodies used for PLA included MCM5 (Proteintech, Chicago, IL, USA) and cleaved‐Notch1 (Immunoway, plano, TX, USA).

Techniques: Modification, Staining, Injection, Quantitative RT-PCR, Transwell Assay, Two Tailed Test

Journal: Advanced Science

Article Title: m 6 A‐Dependent Modulation via IGF2BP3/MCM5/Notch Axis Promotes Partial EMT and LUAD Metastasis

doi: 10.1002/advs.202206744

Figure Lengend Snippet: MCM5 inhibits SIRT1‐mediated NICD1 degradation. A–C) WB analysis determining NICD1 expression or NICD1 stability in indicated cells. D,E) Co‐IP assays examining the effects of MCM5 on NICD1 polyubiquitination or acetylation (AcK) in the presence of MG132. F) Schematic diagram illustrates full‐length (FL) or truncated NICD1 (NICD1‐1‐4). G) Co‐IP analysis of the interaction of MCM5 or SIRT1 with indicated NICD1 truncates. H) The effects of MCM5 on the binding between SIRT1 and NICD1. I) The reversing effect of SIRT1 inhibitor nicotinamide (NAM) on NICD1 acetylation. J,K) The effects of SIRT1 over‐expression or activation by its agonist SRT2183 (J), or SIRT1 inhibition (K) on NICD1 expression. All experiments were repeated three times with similar results.

Article Snippet: The primary antibodies used for PLA included MCM5 (Proteintech, Chicago, IL, USA) and cleaved‐Notch1 (Immunoway, plano, TX, USA).

Techniques: Expressing, Co-Immunoprecipitation Assay, Binding Assay, Over Expression, Activation Assay, Inhibition

Journal: Advanced Science

Article Title: m 6 A‐Dependent Modulation via IGF2BP3/MCM5/Notch Axis Promotes Partial EMT and LUAD Metastasis

doi: 10.1002/advs.202206744

Figure Lengend Snippet: Clinical significance of the IGF2BP3/MCM5/Notch axis in metastatic LUAD. A) qRT‐PCR analysis of MCM5 mRNA in LUAD primary tumors derived from 5 non‐metastatic (NO) and 5 metastatic (YES) patients. (B) IHC of MCM5 expression in primary LUAD tumors and matched brain metastases. n = 10. C,D)Overall survival (OS) analysis of 99 LUAD patients in our own cohort and indicated public LUAD cohorts. E) The expression association between IGF2BP3, MCM5, and NICD1 in LUAD tissues from 50 patients. F) Analysis of epithelial (Keratin) and mesenchymal (Vimentin) markers in circulating tumor cells (CTCs) and bone metastases. Scale bar: 50 µm. G–J) Correlation analysis of IGF2BP3 and p‐EMT plasticity, AT2‐like and HPC state signatures based on the TCGA LUAD datasets using GSEA (G) or GEPIA (H–J). K) Schematic diagram of the IGF2BP3‐MCM5‐Notch axis. Scale bars, 50 µm in (B) and (E). Statistical analyses were performed using a two‐tailed Student's t ‐test (A) and (B) or chi‐squared test (E), *: p < 0.05, ***: p < 0.001.

Article Snippet: The primary antibodies used for PLA included MCM5 (Proteintech, Chicago, IL, USA) and cleaved‐Notch1 (Immunoway, plano, TX, USA).

Techniques: Quantitative RT-PCR, Derivative Assay, Expressing, Two Tailed Test