Journal: Frontiers in Immunology

Article Title: Active LXR signaling, coupled with elevated mitochondrial and glycolytic metabolism contributes to GM-CSF–induced trained immunity

doi: 10.3389/fimmu.2025.1685796

Figure Lengend Snippet: GM-CSF–induced trained immunity depends on mitochondrial metabolism, acetyl-CoA production, and histone acetylation. Monocytes were pretreated with Etomoxir (CPT-1a inhibitor), BPTES (glutaminase inhibitor), or UK5099 (mitochondrial pyruvate carrier inhibitor) 1 h before GM-CSF stimulation. Cells were incubated for 24 h, rested for 5 days, and restimulated with LPS (10 ng/mL) for 24 h. IL-6 and TNFα levels were measured in the supernatants (A–F) . Cells were trained as described previously, rested for 5 days, and subsequently lysed for analysis of gene expression associated with lipid metabolism (G–N) . For histone acetylation analysis, monocytes were primed with GM-CSF or M-CSF for 24 h, rested for 5 days, and lysed on day 6 for ChIP assays using an anti-H3K27ac antibody or control IgG. qPCR was performed on IL6 and TNF promoter regions (O–P) . Statistical analysis was performed using the Wilcoxon matched-pairs signed-rank test. Data represent mean ± SD of at least six donors from three independent experiments. p < 0.05, p < 0.01, p < 0.001.

Article Snippet: Monocytes were cultured at a density of 1 x 10 6 cells/well, 200,000 cells/well or 50,000 cells/well in a 6-, 24- or 96-well plate (Greiner Bio-OneTM) respectively and primed with 1,000 U/ml rhGM-CSF (ImmonoTools, #11343125) or 30 ng/ml rhM-CSF (ImmonoTools, #11343115) in the presence or absence of 1 μM GW3965 (Cayman, #10054) or 1 μM T0901317 (T13) (#HY-10626 MCE), 2.5 μM GSK2033 (LXR antagonist, Cayman, #25443), 10 μM 3PO (PFKFB3 inhibitor, Axonmedchem, #2175), 10 mM 2-DG (#HY-13966, CME), 10 μM (OSI-27) (HY-10423, MCE), 10 μM KC7F2 (HIF1α inhibitor, Medchemexpress, #HY-18777), 50 μM UK5099 (mitochondrial pyruvate carrier (MPC) inhibitor, Medchemexpress, #HY-15475), 40 μM etomoxir (CPT-1a inhibitor, Medchemexpress, #HY-50202), 40 μM BPTES (Glutaminase inhibitor, Medchemexpress, HY-12683) or vehicle for 24 h in RPMI supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate solution, 2 mM L-glutamine solution and 1% penicillin/streptomycin.

Techniques: Incubation, Gene Expression, Control

Journal: The FASEB Journal

Article Title: Pyruvate prevents the onset of the cachectic features and metabolic alterations in myotubes downregulating STAT3 signaling

doi: 10.1096/fj.202200848r

Figure Lengend Snippet: FIGURE 5 Myotubes treated with the MPC inhibitor UK5099 acquire cachectic features. Myotubes have been treated with UK5099 (10 μM final) for 24 h. (A) Representative images of control and UK5099-treated myotubes. (B) Myotube width obtained by ImageJ and calculated in at least 10 randomly chosen fields. (C) Ubiquitin immunoblot. (D) LC3II immunoblot. In (C) and (D) the bar graph reports the mean value of each sample obtained by the ratio with the PVDF membrane used for normalization. (E) Lactate assay. (F) Oxygen consumption rate (OCR). (G) Analysis of mitochondrial membrane potential by confocal microscopy. Mitochondria are stained with TMRM probe (red fluorescence), while blue fluorescence shows the nuclei. The bar graph reports the mean value of fluorescence for each sample measured using ImageJ software in at least 10 randomly chosen fields. (H) PDH activity. C, control myotubes. n = 3; *p < .05.

Article Snippet: Unless otherwise specified, all used reagents were obtained from Sigma- Aldrich, Inc. (St. Louis, MO, USA); SDS- PAGE materials and ECL detection reagents were purchased from Bio- Rad Laboratories, (Hercules, USA); anti- Fbx32/ Atrogin1 (ab168372), anti- OXPHOS (ab110413), antiSTAT3 (ab68153), and anti- Myosin Heavy Chain (MHC) (ab 91506) primary antibodies were from Abcam (Cambridge, UK); anti- PDH- E1 (sc- 377092) and anti- ubiquitin (sc8017) primary antibodies, mitochondrial pyruvate carrier (MPC) inhibitor UK5099 (sc- 361394) and STAT3 inhibitor WP1066 (sc- 203282) were from Santa Cruz Biotechnology (Dallas, TX, USA); anti- LC3B (#3868) and anti- phosphoTyr705- STAT3 (#9145) primary antibodies were from Cell Signaling Technology Inc. (Danvers, MA,USA); Tetramethyl- rhodamine methyl ester (TMRM) probe was from Molecular Probe (Eugene, OR, USA); K- LATE kit for lactate assay was from Megazyme (Bray, Ireland); pyridine and N- tert- Butyldimethylsilyl- N- methyltrifluoroacetamide with 1% tert- Butyldimethylchlorosilane (MBTSTFA + 1% TBDMCS) were from Pierce, ThermoFisher Scientific (Waltham, MA, USA).

Techniques: Control, Ubiquitin Proteomics, Western Blot, Membrane, Lactate Assay, Confocal Microscopy, Staining, Fluorescence, Software, Activity Assay