Journal: Frontiers in Immunology

Article Title: GDF15 orchestrates mitochondrial-immune crosstalk via SMAD7-HIF-1α-PKM2 cascade to attenuate septic liver injury

doi: 10.3389/fimmu.2025.1712741

Figure Lengend Snippet: GDF15 preserves mitochondrial homeostasis in LPS-stimulated macrophages through dual regulation of SMAD7 and PKM2 pathways. (A) HIF-1α and SMAD7 expression in RAW264.7 macrophages across conditions: Untreated, LPS, LPS with rAAV8-mGdf15 overexpression (LPS+GDF15), and LPS with GDF15 knockdown (si-GDF15). β-actin: loading control.(HIF-1α suppression and SMAD7 induction by GDF15.) (B) Cytosolic and nuclear PKM2 protein levels. Lamin B1 (nuclear) and α-tubulin (cytosolic) markers validate fractionation efficiency. Study groups and individual replicates are identified in the figure key.(PKM2 subcellular redistribution modulated by GDF15.) (C) Immunofluorescence of PKM2 (red) and nuclei (DAPI, blue). Arrows indicate nuclear PKM2 accumulation. Scale bar: 15 μm.(Nuclear PKM2 enrichment upon LPS challenge mitigated by GDF15 and exacerbated by GDF15 knockdown.).

Article Snippet: Pharmacological agents included: HIF-1α inhibitor BAY 87-2243 [MedChemExpress, CAS 1227158-85-1; a potent and selective inhibitor of mitochondrial complex I that effectively suppresses HIF-1α protein accumulation under normoxic and hypoxic conditions ( )], PKM2 inhibitor Shikonin [MedChemExpress, CAS 54952-43-1; a specific inhibitor that binds to the PKM2 subunit, suppressing its enzymatic activity and nuclear translocation, with well-documented anti-inflammatory effects ( )], and SMAD7 activator Asiaticoside (MedChemExpress, 16830-15-2; a triterpenoid compound known to upregulate SMAD7 expression and ameliorate inflammation in macrophage models ( )).

Techniques: Expressing, Over Expression, Knockdown, Control, Fractionation, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: GDF15 orchestrates mitochondrial-immune crosstalk via SMAD7-HIF-1α-PKM2 cascade to attenuate septic liver injury

doi: 10.3389/fimmu.2025.1712741

Figure Lengend Snippet: SMAD7 activation suppresses HIF-1α to mediate GDF15-dependent mitochondrial protection in LPS-challenged macrophages. (A) Pharmacological SMAD7 activation by Asiaticoside (20 μM, 48 h). β-actin: loading control. (B) HIF-1α expression under LPS challenge: LPS alone, LPS + AVV-GDF15, or LPS + SMAD7 activation (Asiaticoside). β-actin: loading control. (C) HIF-1α modulation across conditions: LPS, LPS + si-GDF15, LPS + Asiaticoside, or LPS + si-GDF15 + Asiaticoside. β-actin: loading control.

Article Snippet: Pharmacological agents included: HIF-1α inhibitor BAY 87-2243 [MedChemExpress, CAS 1227158-85-1; a potent and selective inhibitor of mitochondrial complex I that effectively suppresses HIF-1α protein accumulation under normoxic and hypoxic conditions ( )], PKM2 inhibitor Shikonin [MedChemExpress, CAS 54952-43-1; a specific inhibitor that binds to the PKM2 subunit, suppressing its enzymatic activity and nuclear translocation, with well-documented anti-inflammatory effects ( )], and SMAD7 activator Asiaticoside (MedChemExpress, 16830-15-2; a triterpenoid compound known to upregulate SMAD7 expression and ameliorate inflammation in macrophage models ( )).

Techniques: Activation Assay, Control, Expressing

Journal: Cell Death & Disease

Article Title: IGF2BP3 regulates EMP1 stability in an m 6 A-dependent manner and activates the TGF-β pathway to promote pancreatic cancer invasion

doi: 10.1038/s41419-025-08155-1

Figure Lengend Snippet: Immunoprecipitation assays obtain silver-stained bands of proteins binding to EMP1-FLAG ( A ); and LC-MS/MS analysis to demonstrate the binding sites of EMP1 and VASP ( B ); In PANC-1 and AsPC-1, Co-IP experiments depict the interactions between EMP1 and VASP ( C ); Immunofluorescence staining of PANC-1 and AsPC-1 demonstrating colocalization of EMP1 and VASP , red ( VASP ), green ( EMP1 ) and blue ( DAPI ) ( D ); Analysis of IGF2BP3 , EMP1 , with VASP correlation in the TCGA-PAAD dataset ( E , F ); Diagram of docking patterns of VASP and SMAD7 proteins ( G ); Immunofluorescence staining of PANC-1 and AsPC-1 demonstrating colocalization of VASP and SMAD7 , red ( VASP ), green ( SMAD7 ) and blue ( DAPI ) ( H ); In PANC-1 and AsPC-1, Co-IP demonstrates the interactions of VASP and SMAD7 ( I) and EMP1 expression affects this binding relation ( J); Western blot detection of EMP1-VASP-SMAD7 axis regulation of TGF-β/Smads signaling pathway in PANC-1 and AsPC-1 ( K) . Scale: 20 μm. ***, P < 0.001.

Article Snippet: Membranes were then incubated with IGF2BP3 (Abcam, ab179807, US), GAPDH (Proteintech, 60004-1-Ig, China), EMP1 (Abcam, ab230445, US), VASP (Proteintech,13472-1-AP, China), SMAD2 (Proteintech,12570-1-AP, China), SMAD3 (Proteintech,66516-1-1 g, China), SMAD7 (Proteintech,25840-1-AP, China), phospho-SMAD2 (CST,138D4, US), and phospho-SMAD3 (CST, 9520S, US) at 4 °C overnight.

Techniques: Immunoprecipitation, Staining, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay, Immunofluorescence, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: IGF2BP3 regulates EMP1 stability in an m 6 A-dependent manner and activates the TGF-β pathway to promote pancreatic cancer invasion

doi: 10.1038/s41419-025-08155-1

Figure Lengend Snippet: METTL3 mediates the modification of m 6 A on the EMP1 RAN, and IGF2BP3 promotes the stabilization of EMP1 RNA based on the recognition of the modification site of m 6 A on EMP1 , and elevated EMP1 protein levels, which increased EMP1 and VASP binding. Meanwhile, the combination of EMP1 and VASP enhanced the function of VASP , which is to impede the inhibition of the pathway of TGF-β signaling by SMAD7 , ultimately leading to the sustained activation of TGF-β in pancreatic cancer. And the effect of METTL3/IGF2BP3/EMP1 on the microenvironment of pancreatic cancer was also demonstrated, including: ( CD8 + T cell, CD68+ Macrophage, α-SMA+ fibroblast, CD20 + B cell and CD11c+ Dendritic cell).

Article Snippet: Membranes were then incubated with IGF2BP3 (Abcam, ab179807, US), GAPDH (Proteintech, 60004-1-Ig, China), EMP1 (Abcam, ab230445, US), VASP (Proteintech,13472-1-AP, China), SMAD2 (Proteintech,12570-1-AP, China), SMAD3 (Proteintech,66516-1-1 g, China), SMAD7 (Proteintech,25840-1-AP, China), phospho-SMAD2 (CST,138D4, US), and phospho-SMAD3 (CST, 9520S, US) at 4 °C overnight.

Techniques: Modification, Binding Assay, Inhibition, Activation Assay