Journal: Nature Communications

Article Title: 6-Phosphogluconate dehydrogenase promotes mitochondrial fusion and immune suppression in tumor-associated monocytic suppressor cells

doi: 10.1038/s41467-025-68102-8

Figure Lengend Snippet: A MDSCs were generated in vitro using IL-6 (40 ng/mL) and GM-CSF (40 ng/mL) from bone marrow (BM) of Pgd fl/fl and Pgd fl/fl LysM Cre mice or wild type (WT) mice with 6AN (5 µM) or vehicle. Sorted MDSCs on day 4 were analyzed for 1318 signaling target proteins by Phospho-Explorer Antibody Array (Full moon BioSystem). B Oxidative PPP checkpoints were blocked by shRNA in WT BM-derived MDSCs. Phospho-IRS-1 S307 levels were examined on sorted M-MDSCs. Blots were processed parallel to samples from the same experiment. Results are representative of two repeats. C 6PG (10, 100, 1000 µM) was added to MDSC cell free lysates for 30 min and IRS-1 S307 phosphorylation measured by western blot. Blots were processed parallel to samples from the same experiment. Results are representative of two repeats. D , E Immunoprecipitated JNK1 and IRS-1 (substrate) were incubated with increasing 6PG concentrations (10, 100, 1000 µM) in an in vitro kinase assay ( D ). In the same experiment, JNK1-IRS-1 binding was evaluated by co-immunoprecipitation (Co-IP) using anti-JNK1 ( E ). Blots were processed parallel to samples from the same experiment. Results are representative of two repeats. F Serine to Alanine (S307A) site-specific mutation in IRS-1 was conducted in M-MDSCs (generated as in ( A )). Flowcytometry-sorted M-MDSCs were co-cultured with anti-CD3/anti-CD28 activated T cells (CFSE-labeled) at a 1:8 (M-MDSC: T cell) ratio. T cell proliferation was evaluated at 72 h. n = 3; One-way ANOVA. G , H M-MDSCs were generated as in ( A , F ). Mitochondrial ROS (mROS) production ( G ) and Arginase 1 expression ( H ) was examined on M-MDSC by flow cytometry. n = 3; One-way ANOVA. I Sorted M-MDSCs were prepared as in ( A , F ). Levels of PI3K (top), AKT (middle) and Drp1 S616 (bottom) phosphorylation were determined by western blot analysis. Blots were processed parallel to samples from the same experiment. Result is representative of two repeats. J – L MDSCs were generated as in ( A , F ), with N-Acetylcysteine (NAC: 0.5 mM) or vehicle (PBS). mROS production in M-MDSCs ( J ), suppressive capacity in co-culture with T cells ( K ) and Arginase expression levels ( L ) determined. n = 3; One-way ANOVA. All data are shown as mean ± SEM.

Article Snippet: The IRS-1 sequence containing a N-terminal HA tag was obtained by PCR using mouse IRS-1 cDNA as a template and cloned into the appropriate multiple cloning sites of the expression vector pcDNA 3.1(+) (CUSABIO).

Techniques: Generated, In Vitro, Ab Array, shRNA, Derivative Assay, Phospho-proteomics, Western Blot, Immunoprecipitation, Incubation, Kinase Assay, Binding Assay, Co-Immunoprecipitation Assay, Mutagenesis, Cell Culture, Labeling, Expressing, Flow Cytometry, Co-Culture Assay