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
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