Journal: Clinical and Translational Medicine

Article Title: FTO‐mediated m 6 A demethylation regulates IGFBP3 expression and AKT activation through IMP3‐dependent P‐body re‐localisation in lung cancer

doi: 10.1002/ctm2.70392

Figure Lengend Snippet: FTO regulates expression of IGFBP3 mRNA in an m 6 A‐dependent manner. (A, B) Quantification of m 6 A and m 6 Am levels in FTO‐kd and FTO‐oe A549 cells. FTO‐kd significantly increased m 6 A ( p <.0001, n = 3) and m 6 Am ( p <.01, n = 3) levels, while FTO‐oe decreased their levels ( p <.0001, n = 3), ( p <.01, n = 3). (C) Dot blots analysis confirmed the increased m 6 A in FTO‐kd A549 cells. (D) ELISA‐based quantification of m 6 A levels in RNA from lung cancer patient tissues. Total m 6 A levels were significantly lower in tumour tissues compared to adjacent tissues ( p <.0001, n = 26). (E, F) Knockdown of m 6 A methyltransferases (Mettl3 and Mettl14) rescued the reduced proliferation caused by FTO‐kd, as shown by CCK8 assays ( p <.0001, n = 10). (G) Heat map analysis of differentially expressed genes based on RNA‐seq in FTO‐kd A549 cells vs. control cells ( n = 3). (H) KEGG pathway analysis of RNA‐seq data identified significant enrichment in the insulin signalling pathway in FTO‐kd A549 cells. (I) Integration of RNA‐seq and MeRIP‐seq data identified IGFBP3 as a key downstream target of FTO. (J) Integrative Genomics Viewer (IGV) visualisation of m 6 A modification sites on IGFBP3 mRNA showed increased m 6 A enrichment near the 3′UTR in FTO‐kd cells. (K, L) qPCR and Western blot analyses confirmed that IGFBP3 mRNA and protein levels were significantly reduced in FTO‐kd cells ( n = 3). (M) IHC analysis of lung tissues from Kras G12D ; FTO wt and Kras G12D ; FTO fl/fl mice demonstrated that FTO negatively regulates IGFBP3 protein expression. (N) Cells ectopically expressing wild‐type FTO or FTO mutants with partial (R316Q) or complete (R316Q/R322Q) loss of catalytic activity showed that FTO's regulation of IGFBP3 expression depends on its m 6 A demethylase activity ( n = 3). (O) Identification of 10 potential m 6 A sites on the IGFBP3 transcript using SRAMP, m 6 AVar, and MeRIP‐seq data. (P, Q) MeRIP‐qPCR confirmed that both above ten sites existed m 6 A, and sites 2, 4, 5, 6, and 7 were regulated by FTO. FTO‐kd increased m 6 A at these sites ( n = 3).

Article Snippet: The membranes were blocked with 5% skim milk in 37°C and then incubated overnight at 4°C with primary antibodies against human FTO (Proteintech, China, 27226‐1‐AP), IGFBP3 (CST, USA, 64143), AKT (CST, USA, 9272), and phosphorylated (p)‐AKT (S473) (CST, USA, 4060), p‐AKT (T308) (CST, USA, 13038), GAPDH (Abcam, UK, ab181602), β‐actin (Abcam, UK, ab8227), Mettl3 (Proteintech, China, 15073‐1AP), Mettl14 (Proteintech, China, 26158‐1AP), IMP3 (Proteintech, China, 14642‐1AP), IMP2 (Proteintech, China, 22803‐1AP), IMP1 (Proteintech, China, 11601‐1AP), YTHDF1 (Proteintech, China, 17474‐1AP), YTHDF2 (Proteintech, China, 24744‐1AP), YTHDF3 (Proteintech, China, 25537‐1AP), YTHDC1 (Proteintech, China, 14392‐1AP), PATL1 (Proteintech, China, 21631‐1AP), LSM14A (Proteintech, China, 18336‐1AP), Tubulin (Proteintech, China, 80762‐1RR), Histon H3 (Proteintech, China, 17168‐1AP), PCNA (CST, USA, 13110), Annexin V (CST, USA, 8555).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Knockdown, RNA Sequencing, Control, Modification, Western Blot, Activity Assay

Journal: Clinical and Translational Medicine

Article Title: FTO‐mediated m 6 A demethylation regulates IGFBP3 expression and AKT activation through IMP3‐dependent P‐body re‐localisation in lung cancer

doi: 10.1002/ctm2.70392

Figure Lengend Snippet: IGFBP3 acts as a functional downstream gene of FTO through regulating the activation of AKT pathway. (A) qPCR analysis of FTO ( p <.0001, n = 36) and IGFBP3 ( p = .0545, n = 36) mRNA expression in tumour and adjacent tissues from 36 lung adenocarcinoma patients. Heatmap analysis showed a positive correlation between FTO and IGFBP3 transcript levels ( r = .6392, p <.0001). (B) Western blot analysis confirmed a significant increase in IGFBP3 protein levels in tumour tissues compared to adjacent tissues ( p <.01, n = 10). (C, D) CCK8 ( p <.0001, n = 10) and colony formation ( p <.0001, n = 6) assays demonstrated that combined knockdown of FTO and IGFBP3 resulted in a more pronounced reduction in cell proliferation compared to FTO‐kd or IGFBP3‐kd alone. IGFBP3‐oe rescued the proliferation defect caused by FTO‐kd. (E) In vivo subcutaneous tumour models using FTO‐kd, IGFBP3‐kd, and FTO‐kd + IGFBP3‐oe cells demonstrated that IGFBP3‐oe reversed the reduction in tumour growth caused by FTO‐kd ( p <.001, n = 6). (F) Western blot analysis showed that IGFBP3‐oe enhanced AKT phosphorylation at S473 and T308 ( n = 3). (G, H) EdU ( p <.0001, n = 6) and CCK8 assays ( p <.0001, n = 10) demonstrated that IGFBP3‐oe promoted cell proliferation, while the AKT inhibitor Capivasertib (10 nM) partially reversed this effect. (I) UMAP‐based dimensionality reduction analysis after scRNA‐sequencing ( n = 2) between Kras G12D ; FTO wt and Kras G12D ; FTO kd . (J) Pseudotime trajectory analysis showed the expression of IGFBP3 and AKT genes along the temporal dimension.

Article Snippet: The membranes were blocked with 5% skim milk in 37°C and then incubated overnight at 4°C with primary antibodies against human FTO (Proteintech, China, 27226‐1‐AP), IGFBP3 (CST, USA, 64143), AKT (CST, USA, 9272), and phosphorylated (p)‐AKT (S473) (CST, USA, 4060), p‐AKT (T308) (CST, USA, 13038), GAPDH (Abcam, UK, ab181602), β‐actin (Abcam, UK, ab8227), Mettl3 (Proteintech, China, 15073‐1AP), Mettl14 (Proteintech, China, 26158‐1AP), IMP3 (Proteintech, China, 14642‐1AP), IMP2 (Proteintech, China, 22803‐1AP), IMP1 (Proteintech, China, 11601‐1AP), YTHDF1 (Proteintech, China, 17474‐1AP), YTHDF2 (Proteintech, China, 24744‐1AP), YTHDF3 (Proteintech, China, 25537‐1AP), YTHDC1 (Proteintech, China, 14392‐1AP), PATL1 (Proteintech, China, 21631‐1AP), LSM14A (Proteintech, China, 18336‐1AP), Tubulin (Proteintech, China, 80762‐1RR), Histon H3 (Proteintech, China, 17168‐1AP), PCNA (CST, USA, 13110), Annexin V (CST, USA, 8555).

Techniques: Functional Assay, Activation Assay, Expressing, Western Blot, Knockdown, In Vivo, Phospho-proteomics, Sequencing

Journal: Clinical and Translational Medicine

Article Title: FTO‐mediated m 6 A demethylation regulates IGFBP3 expression and AKT activation through IMP3‐dependent P‐body re‐localisation in lung cancer

doi: 10.1002/ctm2.70392

Figure Lengend Snippet: IMP3 inhibits the translation efficiency of IGFBP3 mRNA in an m 6 A‐dependent manner. (A) Products of RNA‐pulldown experiments with biotinylated RNA targeting m 6 A positions 4–7 were analysed by silver staining. (B) Western blot analysis after RNA‐pulldown assays using biotinylated RNA probes targeting m 6 A sites 4–7 of IGFBP3 showed that YTHDC1 and IMP3 bind to these sites in A549 and PC9 cells. (C) qPCR after RNA Binding Protein Immunoprecipitation (RIP) assays with anti‐YTHDC1 and anti‐IMP3 antibodies confirmed their binding to m 6 A‐modified sites in the 3′UTR of IGFBP3 mRNA ( n = 3). (D) RIP‐qPCR analysis showed that FTO‐kd significantly increased the binding of YTHDC1 and IMP3 to IGFBP3 mRNA in an m 6 A‐dependent manner. (E) The immunofluorescence co‐localisation analysis indicated that FTO‐kd enhance the co‐localisation signals of IMP3 and IGFBP3 ( p <.001, n = 3). (F, G) RNA‐pulldown assays using six biotinylated RNA fragments of IGFBP3 revealed that YTHDC1 and IMP3 bind specifically to the wild‐type 3′UTR but not to mutated or unmodified 3′UTR fragments. (H) The schematic showed the manner in which the sucrose density gradient separates polyribosomes. (I–K) Ribosome profiling showed that FTO‐kd decreased the translation efficiency (TE) of IGFBP3 mRNA ( n = 3). (L, M) TE analysis of IGFBP3 following knockdown of IMP3 or YTHDC1. IMP3‐kd significantly enhanced IGFBP3 TE ( p <.001, n = 3), while YTHDC1‐kd had no effect ( p = .305, n = 3). (N) GFP‐tagged IGFBP3 plasmids transfected into IMP3‐kd and control A549 cells demonstrated that IMP3 inhibits IGFBP3 TE, as measured by GFP fluorescence ( p <.001, n = 6). (O) Absolute quantification of IGFBP3 transcript copy number and TE in IMP3‐kd and control cells ( p <.01, n = 4).

Article Snippet: The membranes were blocked with 5% skim milk in 37°C and then incubated overnight at 4°C with primary antibodies against human FTO (Proteintech, China, 27226‐1‐AP), IGFBP3 (CST, USA, 64143), AKT (CST, USA, 9272), and phosphorylated (p)‐AKT (S473) (CST, USA, 4060), p‐AKT (T308) (CST, USA, 13038), GAPDH (Abcam, UK, ab181602), β‐actin (Abcam, UK, ab8227), Mettl3 (Proteintech, China, 15073‐1AP), Mettl14 (Proteintech, China, 26158‐1AP), IMP3 (Proteintech, China, 14642‐1AP), IMP2 (Proteintech, China, 22803‐1AP), IMP1 (Proteintech, China, 11601‐1AP), YTHDF1 (Proteintech, China, 17474‐1AP), YTHDF2 (Proteintech, China, 24744‐1AP), YTHDF3 (Proteintech, China, 25537‐1AP), YTHDC1 (Proteintech, China, 14392‐1AP), PATL1 (Proteintech, China, 21631‐1AP), LSM14A (Proteintech, China, 18336‐1AP), Tubulin (Proteintech, China, 80762‐1RR), Histon H3 (Proteintech, China, 17168‐1AP), PCNA (CST, USA, 13110), Annexin V (CST, USA, 8555).

Techniques: Silver Staining, Western Blot, RNA Binding Assay, Immunoprecipitation, Binding Assay, Modification, Immunofluorescence, Knockdown, Transfection, Control, Fluorescence, Quantitative Proteomics

Journal: Clinical and Translational Medicine

Article Title: FTO‐mediated m 6 A demethylation regulates IGFBP3 expression and AKT activation through IMP3‐dependent P‐body re‐localisation in lung cancer

doi: 10.1002/ctm2.70392

Figure Lengend Snippet: IMP3 inhibits IGFBP3 mRNA translation by promoting its localisation to P‐bodies. (A) Dot blot assay using an anti‐m 6 A antibody indicates significantly lower m 6 A modifications in polysomal fractions compared to other cytoplasmic components ( n = 3). (B) Immunoprecipitation (IP) assay with anti‐PATL1 antibody reveals that IMP3 co‐localises with the P‐body marker LSM14A. (C) RIP‐qPCR analysis shows decreased localisation of IGFBP3 mRNA to P‐bodies upon IMP3‐kd ( p <.001, n = 3), while (D) demonstrates increased P‐body localisation of IGFBP3 mRNA upon IMP3‐oe ( p <.01, n = 3). (E) Schematic representation of the tethering experiment using the MCP‐IMP3 system with Rluc‐MS2 reporter mRNAs to assess IMP3's regulatory role on translation. (F) Western blot analysis confirms the expression of FLAG‐MCP (negative control) and FLAG‐IMP3‐MCP proteins, ensuring consistent transfection efficiency across samples (see Figure ). (G) Tethering of FLAG‐IMP3‐MCP to reporter mRNA results in a ∼45% reduction in luciferase activity ( p <.001, n = 3). (H–J) Schematic representation of the method used to fluorescently label P‐bodies in the A549 cell line using GFP‐LSM14A, contrasting with a control expressing a non‐localising truncated version (GFP‐LSM14A‐Δ). Flow particle sorting (FACS) distinguishes GFP‐LSM14A‐labelled P‐bodies from non‐P‐body particles based on size and fluorescence. (K) Dot blot quantification revealed that m 6 A levels in P‐bodies are significantly higher than in non‐P‐body fractions ( n = 3). (L) IMP3‐kd leaded to a significant reduction in IGFBP3 enrichment in P‐bodies ( p <.01, n = 3). (M) FISH analysis demonstrates that IGFBP3 mRNA co‐localises with P‐bodies more frequently than ADSL mRNA, as evidenced by confocal microscopy ( p <.0001, n = 3). (N) IMP3‐kd diminished the co‐localisation of IGFBP3 mRNA with P‐bodies ( p <.0001, n = 3).

Article Snippet: The membranes were blocked with 5% skim milk in 37°C and then incubated overnight at 4°C with primary antibodies against human FTO (Proteintech, China, 27226‐1‐AP), IGFBP3 (CST, USA, 64143), AKT (CST, USA, 9272), and phosphorylated (p)‐AKT (S473) (CST, USA, 4060), p‐AKT (T308) (CST, USA, 13038), GAPDH (Abcam, UK, ab181602), β‐actin (Abcam, UK, ab8227), Mettl3 (Proteintech, China, 15073‐1AP), Mettl14 (Proteintech, China, 26158‐1AP), IMP3 (Proteintech, China, 14642‐1AP), IMP2 (Proteintech, China, 22803‐1AP), IMP1 (Proteintech, China, 11601‐1AP), YTHDF1 (Proteintech, China, 17474‐1AP), YTHDF2 (Proteintech, China, 24744‐1AP), YTHDF3 (Proteintech, China, 25537‐1AP), YTHDC1 (Proteintech, China, 14392‐1AP), PATL1 (Proteintech, China, 21631‐1AP), LSM14A (Proteintech, China, 18336‐1AP), Tubulin (Proteintech, China, 80762‐1RR), Histon H3 (Proteintech, China, 17168‐1AP), PCNA (CST, USA, 13110), Annexin V (CST, USA, 8555).

Techniques: Dot Blot, Immunoprecipitation, Marker, Western Blot, Expressing, Negative Control, Transfection, Luciferase, Activity Assay, Control, Fluorescence, Confocal Microscopy

Journal: Clinical and Translational Medicine

Article Title: FTO‐mediated m 6 A demethylation regulates IGFBP3 expression and AKT activation through IMP3‐dependent P‐body re‐localisation in lung cancer

doi: 10.1002/ctm2.70392

Figure Lengend Snippet: Proposed working model for promotive effects of FTO on NSCLC progression. This work finds that FTO‐mediated modulation of m 6 A and effects on IGFBP3 translation play critical oncogenic roles in LUAD. Our results suggest that targeting of the FTO–IGFBP3–AKT axis may be a promising strategy for the treatment of LUAD.

Article Snippet: The membranes were blocked with 5% skim milk in 37°C and then incubated overnight at 4°C with primary antibodies against human FTO (Proteintech, China, 27226‐1‐AP), IGFBP3 (CST, USA, 64143), AKT (CST, USA, 9272), and phosphorylated (p)‐AKT (S473) (CST, USA, 4060), p‐AKT (T308) (CST, USA, 13038), GAPDH (Abcam, UK, ab181602), β‐actin (Abcam, UK, ab8227), Mettl3 (Proteintech, China, 15073‐1AP), Mettl14 (Proteintech, China, 26158‐1AP), IMP3 (Proteintech, China, 14642‐1AP), IMP2 (Proteintech, China, 22803‐1AP), IMP1 (Proteintech, China, 11601‐1AP), YTHDF1 (Proteintech, China, 17474‐1AP), YTHDF2 (Proteintech, China, 24744‐1AP), YTHDF3 (Proteintech, China, 25537‐1AP), YTHDC1 (Proteintech, China, 14392‐1AP), PATL1 (Proteintech, China, 21631‐1AP), LSM14A (Proteintech, China, 18336‐1AP), Tubulin (Proteintech, China, 80762‐1RR), Histon H3 (Proteintech, China, 17168‐1AP), PCNA (CST, USA, 13110), Annexin V (CST, USA, 8555).

Techniques: