sc 203282  (Santa Cruz Biotechnology)

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: Pharmacological research

Article Title: Resistin induces cardiac fibroblast-myofibroblast differentiation through JAK/STAT3 and JNK/c-Jun signaling.

doi: 10.1016/j.phrs.2020.105414

Figure Lengend Snippet: Fig. 5. Resistin overexpression induces fibrosis signaling pathways in adult mice- C57B6 adult mice were infected with AAV9-resistin (AAV9-Retn) or AAV9-empty vectors for 10 weeks. The phosphorylation of JAK2, STAT3 (A) and JNK, c-Jun (B) was analyzed by western blotting with densitometry quantification shown below the blots. β-actin was used as internal controls. The data are mean ± SEM of n = 5. p*< 0.05, **p < 0.01 vs AAV9-Empty.

Article Snippet: For JAK and JNK inhibition, cells were incubated with JAK2 inhibitor (WP1066, # sc-203282, Santa Cruz Biotechnology) and JNK inhibitor (SP600125, # sc-200635, Santa Cruz Biotechnology) at the indicated concentrations selected as per earlier studies [28].

Techniques: Over Expression, Protein-Protein interactions, Infection, Phospho-proteomics, Western Blot

Journal: Pharmacological research

Article Title: Resistin induces cardiac fibroblast-myofibroblast differentiation through JAK/STAT3 and JNK/c-Jun signaling.

doi: 10.1016/j.phrs.2020.105414

Figure Lengend Snippet: Fig. 4. Resistin promotes fibrogenesis via activating JAK-STAT and JNK-c-Jun signaling pathways in cardiac fibroblasts, independently of the TGFβ1 pathway - Cardiac fibroblasts were treated with resistin (100 ng/mL) and TGFβ1 (10 ng/mL) for the indicated times. The phosphorylation of Smad3, JAK2, STAT3, JNK, c-Jun was analyzed by western blotting (A), and densitometry quantifications were performed (B). β-actin was used as internal controls. The data are mean ± SEM of at least three experiments in triplicates. p*< 0.05, **p < 0.01, ***p < 0.001, 0 min vs indicated minutes in figure.

Article Snippet: For JAK and JNK inhibition, cells were incubated with JAK2 inhibitor (WP1066, # sc-203282, Santa Cruz Biotechnology) and JNK inhibitor (SP600125, # sc-200635, Santa Cruz Biotechnology) at the indicated concentrations selected as per earlier studies [28].

Techniques: Protein-Protein interactions, Phospho-proteomics, Western Blot

Journal: Pharmacological research

Article Title: Resistin induces cardiac fibroblast-myofibroblast differentiation through JAK/STAT3 and JNK/c-Jun signaling.

doi: 10.1016/j.phrs.2020.105414

Figure Lengend Snippet: Fig. 6. Resistin fails to induce expression of fibrotic genes in JAK-inhibited and JNK–inhibited cells – A) Cardiac fibroblasts were treated with JAK inhibitor WP1066 (5 μM) for 30 min and stimulated with resistin (100 ng/mL) for an additional 2 h. The phosphorylation of JAK2 and STAT3 was analyzed by western blotting. B) Representative fluorescence microscopic images of cardiac fibroblasts treated with JAK2 inhibitor for 2 h and stimulated with resistin in presence of JAK2 inhibitor for an additional 48 h and then stained for F actin to visualize stress fibers. C) mRNA expression of αSma, Col1a1, Ccn2, Fn, Mmp9, and Timp1 was analyzed by q-PCR in cardiac fibroblasts treated with JAK2 inhibitor for 2 h and stimulated with resistin for an additional 48 h. D) Cardiac fibroblasts were treated with JNK inhibitor SP600125 (25 μM) for 30 min and stimulated with resistin (100 ng/mL) in the presence of JAK2 inhibitor for an additional 2 h. The phosphorylation of JNK and c-Jun was analyzed by western blotting. E) Representative fluorescence microscopic images of cardiac fibroblasts treated with JNK inhibitor for 2 h and stimulated with resistin for an additional 48 h and then stained for F actin to visualize stress fibers; bar size =200 μM. F) mRNA expression of αSma, Col1a1, Ccn2, Fn, Mmp9, and Timp1 was analyzed by q-PCR in cardiac fibroblasts treated with JAK2 inhibitor for 2 h and stimulated with resistin for additional 48 h. 18S rRNA was used as an internal control. The data are mean ± SEM of three experiments in triplicates, p*< 0.05, ***p < 0.001.

Article Snippet: For JAK and JNK inhibition, cells were incubated with JAK2 inhibitor (WP1066, # sc-203282, Santa Cruz Biotechnology) and JNK inhibitor (SP600125, # sc-200635, Santa Cruz Biotechnology) at the indicated concentrations selected as per earlier studies [28].

Techniques: Expressing, Phospho-proteomics, Western Blot, Fluorescence, Staining, Control

Journal: Pharmacological research

Article Title: Resistin induces cardiac fibroblast-myofibroblast differentiation through JAK/STAT3 and JNK/c-Jun signaling.

doi: 10.1016/j.phrs.2020.105414

Figure Lengend Snippet: Fig. 8. Resistin knockout mice on high-fat diet exhibit lower activation of JAK2 and JNK signaling pathways – Mice were treated as in Fig. 7. A) Protein samples were extracted from heart muscles and analyzed for the total and phosphorylated forms of JAK2, STAT3, JNK, and c-Jun by western blotting. B) Quantification of band densities in (A). β-actin was used as internal control. The data are mean ± SEM of n = 5. p*< 0.05; **p < 0.01. C) Schematic diagram summarizing the role of JAK2 and JNK signaling pathways in resistin-driven regulation of fibroblast-myofibroblast conversion. Resistin potentially binds to TLR4 and activates JAK2 which in turn phosphorylates STAT3 causing it to translocate into the nucleus and activate pro-fibrotic target genes (i.e. Mmp9, Ccn2, Col1a1, vimentin and Tgfβ1). Resistin also activates JNK, potentially through ASK as we demonstrated previously [26], phosphorylating c-Jun causing it to translocate into the nucleus and activate pro-fibrotic target genes (i.e. Ccn2, Col1a1, Fn, Lox and Tgfβ1). TGFβ1 binds to TGFβ1R and activates the profibrotic genes by phosphorylating its downstream targets like Smad3, and JNK.

Article Snippet: For JAK and JNK inhibition, cells were incubated with JAK2 inhibitor (WP1066, # sc-203282, Santa Cruz Biotechnology) and JNK inhibitor (SP600125, # sc-200635, Santa Cruz Biotechnology) at the indicated concentrations selected as per earlier studies [28].

Techniques: Knock-Out, Activation Assay, Protein-Protein interactions, Muscles, Western Blot, Control