Fig 2: The multiplex immunohistochemistry staining demonstrated the distribution of IGFBP3 in the tumor immune microenvironment of PDAC, showing its presence in both cancer-associated fibroblasts and tumor cells.

Fig 3: Spatial transcriptome analysis. (A) Cell clustering. (B) Expression of IGFBP3 across 7 samples. (C) Cell annotation. (D, E) Cell communication analysis. (F) IGF pathway analysis.

Fig 4: IMP3 inhibits the translation efficiency of IGFBP3 mRNA in an m6A‐dependent manner. (A) Products of RNA‐pulldown experiments with biotinylated RNA targeting m6A positions 4–7 were analysed by silver staining. (B) Western blot analysis after RNA‐pulldown assays using biotinylated RNA probes targeting m6A 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 m6A‐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 m6A‐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).

Fig 5: IMP3 inhibits IGFBP3 mRNA translation by promoting its localisation to P‐bodies. (A) Dot blot assay using an anti‐m6A antibody indicates significantly lower m6A 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 S6B). (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 m6A 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).