stat3 inhibitor iii wp1066  (Millipore)

Journal: International journal of molecular sciences

Article Title: STAT3 Signalling Drives LDH Up-Regulation and Adiponectin Down-Regulation in Cachectic Adipocytes.

doi: 10.3390/ijms242216343

Figure Lengend Snippet: Figure 4. STAT3 signalling drives the effects induced by CM CT26. (A) Murine adipocytes were treated with serum-free medium overnight and then stimulated for the indicated time with CM CT26. The levels of total and phosphorylated-Tyr705 of STAT3 were detected using immunoblots. (Panels B–F) show murine adipocytes that were treated with CM CT26 for 48 h. Where indicated, WP1066 (10 µM) was added to CM CT26 or to the control. (B) Representative images of adipocytes. (C) Measures of lipid droplet width and (D) adipocyte area, both calculated using at least ten fields chosen randomly. (E) Lactate assay. (F) Analysis of oxygen consumption reported as oxygen consumption rate (OCR). C, control; CM CT26: conditioned media from colon cancer cell CT26; * p < 0.05; n = 3; scale: 50 µm.

Article Snippet: Unless otherwise specified, all reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA); anti-Adiponectin (ab22554) primary antibody was obtained from Abcam (Cambridge, UK); anti-LDH (sc-33781) and anti-STAT3 (sc-8019) primary antibodies and STAT3 Inhibitor III WP1066 (sc-203282) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); anti-phospho-Tyr705-STAT3 (#9145) primary antibody was purchased from Cell Signalling Technology Inc. (Danvers, MA, USA); SDS-PAGE materials and ECL reagents were obtained from Bio-Rad Laboratories (Hercules, CA, USA); and K-LATE kit for lactate assay was purchased from Megazyme (Bray, Ireland).

Techniques: Western Blot, Control, Lactate Assay

Journal: International journal of molecular sciences

Article Title: STAT3 Signalling Drives LDH Up-Regulation and Adiponectin Down-Regulation in Cachectic Adipocytes.

doi: 10.3390/ijms242216343

Figure Lengend Snippet: Figure 5. STAT3 controls LDH and adiponectin levels. Murine adipocytes were treated with CM CT26 for 48 h. Where indicated, WP1066 (10 µM) was added to CM CT26. (A) LDH and (B) adiponectin levels assayed using immunoblotting. PVDF membranes were used for normalization, and the mean values are reported in the bar graph. C, control; CM CT26: conditioned media from colon cancer cell CT26; LDH: lactate dehydrogenase; acrp30: adiponectin; * p < 0.05; n = 3.

Article Snippet: Unless otherwise specified, all reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA); anti-Adiponectin (ab22554) primary antibody was obtained from Abcam (Cambridge, UK); anti-LDH (sc-33781) and anti-STAT3 (sc-8019) primary antibodies and STAT3 Inhibitor III WP1066 (sc-203282) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); anti-phospho-Tyr705-STAT3 (#9145) primary antibody was purchased from Cell Signalling Technology Inc. (Danvers, MA, USA); SDS-PAGE materials and ECL reagents were obtained from Bio-Rad Laboratories (Hercules, CA, USA); and K-LATE kit for lactate assay was purchased from Megazyme (Bray, Ireland).

Techniques: Western Blot, Control

Journal: International journal of molecular sciences

Article Title: STAT3 Signalling Drives LDH Up-Regulation and Adiponectin Down-Regulation in Cachectic Adipocytes.

doi: 10.3390/ijms242216343

Figure Lengend Snippet: Figure 6. Proposed model of CM CT26 effects in adipocytes. The treatment of adipocytes with CM CT26 induces STAT3 phosphorylation on Tyr 205. The activation of STAT3 cascade induces the increase in lactate production and the decrease in oxygen consumption, associated with the enhanced level of LDH and the concomitant decrease in adiponectin. The blocking of STAT3 cascade using the inhibitor WP1066 hinders the down-stream effects due to CM CT26, which becomes ineffective. CM CT26: conditioned media from colon cancer cell CT26.

Article Snippet: Unless otherwise specified, all reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA); anti-Adiponectin (ab22554) primary antibody was obtained from Abcam (Cambridge, UK); anti-LDH (sc-33781) and anti-STAT3 (sc-8019) primary antibodies and STAT3 Inhibitor III WP1066 (sc-203282) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); anti-phospho-Tyr705-STAT3 (#9145) primary antibody was purchased from Cell Signalling Technology Inc. (Danvers, MA, USA); SDS-PAGE materials and ECL reagents were obtained from Bio-Rad Laboratories (Hercules, CA, USA); and K-LATE kit for lactate assay was purchased from Megazyme (Bray, Ireland).

Techniques: Phospho-proteomics, Activation Assay, Blocking Assay

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 6 STAT3 signaling pathway. (A) Myotubes have been stimulated with CM CT26 for the indicated period and STAT3 phosphorylation (Tyr705) has been analyzed by immunoblot. The bar graph shows the STAT3 phosphorylated level on Tyr705. Myotubes have been treated with CM CT26 for 24 h. Where indicated, the STAT3 inhibitor WP1066 (10 μM) has been added to CM CT26 along the experiment. (B) Representative images of myotubes. (C) Myotube width measured using Image J software in at least 10 randomly chosen fields. The mean value for each sample has been reported in the bar graph. (D) Lactate assay. (E) Oxygen consumption rate (OCR). (F) Myotubes have been stimulated with CM CT26 for the indicated period. Sodium pyruvate (20 mM final) has been added 30 min before the treatment with CM CT26. STAT3 phosphorylation (Tyr705) has been analyzed by immunoblot. The bar graph shows the STAT3 phosphorylated level on Tyr705. C, control myotubes. n = 4; *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: Phospho-proteomics, Western Blot, Software, Lactate Assay, Control