Journal: bioRxiv
Article Title: Tumor-derived Extracellular Vesicles Induce ER Stress to Drive Tolerogenic Dendritic Cell Development in the Tumor Microenvironment
doi: 10.64898/2026.02.10.705213
Figure Lengend Snippet: (A) Flow cytometry analysis of neutral lipid accumulation in splenic DCs treated with PBS (Ctrl), tumor EVs (20 µg/ml; 24h), or tumor EVs (20 µg/ml; 24h) in the presence of the IRE1 α inhibitor 4μ8c. Left , Representative histograms of BODIPY 493/503 fluorescence. Right , quantification of the normalized mean fluorescence intensity (MFI) (n=3). Representative of three independent experiments. (B) GSEA plot from the scRNAseq data in , showing enrichment of the "Regulation of Lipid Metabolism by PPARα" gene set in EV-treated DCs compared to controls. Adjusted p-value (Padj < 0.05) is displayed. Representative of two independent experiments. (C) Western blot analysis of nuclear PPAR-α levels in BMDCs treated with or without EVs (20 µg/ml; 24h). Histone H3 is shown as a nuclear loading control. Representative of three independent experiments. (D) Immunoblot analysis of nuclear PPAR-α protein expression in BMDCs following treatment with PBS (Ctrl), tumor EVs (20 µg/ml; 24h), or EVs in the presence of the IRE1α inhibitor 4μ8c. Lamin B1 serves as the nuclear loading control. Representative of three independent experiments. (E) Quantification of PPAR-α DNA-binding activity in nuclear extracts from BMDCs treated with EVs (20 µg/ml; 24h), 4μ8c, or both (n=4-7). Representative of three independent experiments. (F) Flow cytometry quantification of neutral lipid content (BODIPY 493/503) in DCs treated with EVs (20 µg/ml; 24h) with or without the PPAR-α inhibitor TPST-1120. Representative of three independent experiments. (G) Flow cytometric analysis of fatty acid β-oxidation (FAO) activity in splenic DCs treated with PBS (Ctrl) or EVs (20 µg/ml; 24h) using FAO-Blue (n=5). (H) Normalized mRNA expression of Cpt1a , a key rate-limiting enzyme in FAO and a known PPAR-α target gene, in tumor-infiltrating DCs from the model in , comparing EV-uptaking (Emerald Green + ) versus bystander (Emerald Green - ) DC subsets (n=6). Representative of two independent experiments. (I) Heatmap of relative gene expression (Z-score) for triglyceride lipolysis ( Pnpla2 , Lipe , Mgll ) and fatty acid transport genes ( Fabp5 , Fabp4 ) in EV-uptaking (Exogreen + ) versus bystander (Exogreen - ) LNDCs isolated in vivo . Representative of two independent experiments. (J) Flow cytometric analysis of fatty acid uptake by splenic DCs treated with PBS (Ctrl) or EVs (20 µg/ml; 24h) using TF2-C12. Right , representative histogram. Representative of three independent experiments. All data is reported as mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 based on a one-way ANOVA (A,E,F) and an unpaired Student’s t-test (G,H,J). FAO , fatty acid oxidation; GSEA , gene set enrichment analysis; MFI , mean fluorescence intensity; PPARα , peroxisome proliferator-activated receptor alpha; BMDCs , bone marrow-derived dendritic cells; Ctrl , control ; EVs , extracellular vesicles; IRE1α, inositol-requiring enzyme 1 alpha.
Article Snippet: Primary antibodies included: Rab27a (Cell Signaling Technology [CST], Cat# 69295S), SREBP2 (Novus Biologicals, Cat# NB100-74543), ATF4 (CST, Cat# 11815S), ATF6 (CST, Cat# 65880S), IRE1α (CST, Cat# 3294S), SREBP1 (clone 2A4; Santa Cruz Biotechnology, Cat# sc-13551), Lamin B1 (clone B-10; Santa Cruz, Cat# sc-374015), XBP-1s (CST, Cat# 40435), PPARα (Thermo Fisher Scientific, Cat# PA1-822A), PPARγ (Santa Cruz, Cat# sc-7273), PPARβ (Santa Cruz, Cat# sc-74517) and Histone H3 (Santa Cruz, Cat# sc-517576).
Techniques: Flow Cytometry, Fluorescence, Western Blot, Control, Expressing, Binding Assay, Activity Assay, Gene Expression, Isolation, In Vivo, Derivative Assay
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