Primary hTM cells were cultured on plastic dishes or glass coverslips to confluency (approximately 90%) in 10% fetal bovine serum growth media. Cells were subsequently serum starved for 24 hours, after which respective treatment with vehicle control (veh), LPA (20 µM; catalog number: 10010093; Cayman Chemical, Ann Arbor, MI, USA), IL6 (100 ng/mL; catalog number: SRP3096; Sigma Aldrich, St. Louis, MO, USA)/sIL6R (200 ng/mL; catalog number: SRP3097; Sigma Aldrich, St. Louis, MO, USA), or both (LPA + IL6/sIL6R) in serum-free media was done for 24 hours. In another set of experiments, the aforementioned treatments were performed in the presence or absence of 2 µM verteporfin (YAP inhibitor, without light stimulation using aluminum foil; catalog number: 17334; Cayman Chemical, Ann Arbor, MI, USA) or 2 µM STAT3 inhibitor (Catalog number: 573097; Sigma Aldrich, St. Louis, MO, USA) in serum-free media for 24 hours. The concentration of verteporfin used in this study has previously been verified to be safe and efficacious in hTM cells and other ocular/nonocular cells.57 (link)–59 (link) Herein, we determined a safe and effective dose for the STAT3 inhibitor (that is, 2 µM) by performing a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Supplementary Fig. S1 ) and Western blotting.
Stat3 inhibitor
Manufactured by Merck Group
Sourced in United States
The STAT3 inhibitor is a laboratory tool designed to inhibit the STAT3 (Signal Transducer and Activator of Transcription 3) protein. STAT3 is a transcription factor that plays a role in various cellular processes, including cell growth, differentiation, and survival. This product is intended for use in research applications to study the effects of STAT3 inhibition on cellular function and signaling pathways.
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Lab products found in correlation
Interleukin 6 (il 6), by Merck Group (1 mentions)
Sil6r, by Merck Group (1 mentions)
Verteporfin, by Cayman Chemical (1 mentions)
N ethylmaleimide nem, by Merck Group (1 mentions)
Recombinant human il 6, by R&D Systems (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
L cysteine, by Merck Group (1 mentions)
Sta 21, by Merck Group (1 mentions)
Fitc conjugated annexin 5, by BD (1 mentions)
Actinomycin, by Merck Group (1 mentions)
Apocynin, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (1 mentions)
Cryptotanshinone, by Merck Group (1 mentions)
Cis diammineplatinum 2 dichloride, by Merck Group (1 mentions)
Recombinant human il 6, by Thermo Fisher Scientific (1 mentions)
Recombinant human leptin, by R&D Systems (1 mentions)
96 well plate, by Corning (1 mentions)
Cck 8 reagent, by Beyotime (1 mentions)
Multiskan mk, by Thermo Fisher Scientific (1 mentions)
5 protocols using stat3 inhibitor
1
Modulation of hTM Cell Signaling
2
Platelet Activation Signaling Pathway
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Commercial reagents used in the study included: PL (Cayman Chemical Co., Ann Arbor, MI), the STAT3 inhibitor STA21 (Sigma Aldrich, St. Louis, MO), the JAK2-inhbitor AG490 (InvivoGen, San Diego, CA), the Syk inhibitor SykII (Merck Millipore, Billerica, MA), human recombinant IL-6 (R & D Systems, Minneapolis, MN), the extracellular domain of human IL-6 receptor-α (R & D Systems), Actinomycin (Sigma Aldrich), apocynin (Abcam Biochemicals. Cambridge, MA), free glutathione (GSH, Sigma Aldrich), L-cysteine (Sigma Aldrich), N-ethylmaleimide (NEM, Sigma Aldrich); dithiothreitol (DTT, Sigma Aldrich), fibrillary type I collagen (Helena laboratories, Beaumont, TX) and FITC-conjugated annexin V (BD Bioscience, San Jose, CA). Antibodies used in the study were: a FITC-conjugated monoclonal CD62p (BD Bioscience), a PE-conjugated monoclonal anti-CD42b (BD Bioscience), and antibodies for total and phosphorylated JAK2, STAT3, Syk, and PLCγγ2 (all from Cell Signaling Technologies, Danvers, MA).
3
Oral Cancer Cell Cytotoxicity Assay
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Human oral squamous carcinoma cells HSC‐2 and HSC‐3 were obtained from the Cell Resource Center, Institute of Developmental, Aging and Cancer, Tohoku University (Sendai, Japan). Cells were seeded at a density of 1 × 105 cells/mL and cultured at 37°C in a humidified atmosphere of 95% air and 5% CO2 in DMEM (Wako) supplemented with 10% FBS (Sigma‐Aldrich). Upon reaching sub‐confluence, cells were incubated in culture medium with or without cisplatin [cis‐Diammineplatinum(II)] dichloride (1‐5 μmol/L); Sigma‐Aldrich) for 0‐72 hours. In some experiments, the cells were incubated in culture medium containing cisplatin and fludarabine (10 μmol/L; STAT1 inhibitor; Sigma‐Aldrich) or cryptotanshinone (10 μmol/L; STAT3 inhibitor; Sigma‐Aldrich).
4
Antibody Detection and Neutralization Protocol
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The following primary monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were used to detect proteins by immunofluorescence labeling and immunoblot analysis and/or to neutralize the effects of leptin and IL-6: mouse anti-leptin mAb (44802), anti-leptin receptor mAb (52263) (R&D Systems, Minneapolis, MN, USA), rat anti-IL-6 mAb (MQ2-13A5; Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti-phospho STAT3 (Tyr705) mAb (3E2), rabbit anti-STAT3 pAb (D47E7), anti-SOCS3 pAb (Cell Signaling Technology, Danvers, MA, USA), mouse anti-β-actin mAb (E1C605; EnoGene, New York, NY, USA), rabbit anti-claudin-5 mAb (EPR7583), anti-VE-cadherin pAb (Abcam, Cambridge, MA, USA), rabbit anti-occludin pAb, anti-ZO-1 pAb (Invitrogen, Carlsbad, CA, USA), and mouse anti-D2-40 mAb (413451; Nichirei, Tokyo, Japan). Mouse IgG (11711) isotype controls were purchased from R&D Systems. Recombinant human leptin was obtained from R&D Systems. Recombinant human IL-6 was purchased from Peprotech (Rocky Hill, NJ, USA). STAT3 inhibitor was obtained from EMD Millipore (Billerica, MA, USA).
5
In Vitro VSMC Proliferation Assays
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In vitro cell proliferation assays were performed as described previously [50 (link), 51 (link)]. Briefly, VSMCs (2 × 103/well) were seeded into 96-well plates (Corning, Lowell, MA, USA). Cells were cultured in NG, Mtol and HG media supplemented with quercetagetin (5.5 μmol/L, Pim-1 inhibitor, Merck, Germany), stattic (10 μmol/L, STAT3 inhibitor, Merck) and dimethylsulphoxide (DMSO, ≤0.3%, v/v) for 48 hours. After washing twice with 1×PBS, a total of 100 μL DMEM plus 10 μL CCK-8 reagents (Beyotime Institute of Biotechnology, Jiangsu, China) were added to each well and the optical density (OD) was measured 4 hours later using a microplate reader (Multiskan MKS, Thermo Scientific, MA, USA) at dual wavelength (450/630 nm). Each group was duplicated in six wells and each assay was performed in triplicate.
Cell number was determined as described previously [52 (link)]. Briefly, VSMCs were seeded into 6-well plates (5 × 105 per well). After overnight recovery in an incubator at 37°C/5% CO2, the cells were serum-deprived for 12 hours and exposed to the aforementioned conditions for 48 hours and then counted using a hemocytometer ( n = 3 wells / group).
Cell number was determined as described previously [52 (link)]. Briefly, VSMCs were seeded into 6-well plates (5 × 105 per well). After overnight recovery in an incubator at 37°C/5% CO2, the cells were serum-deprived for 12 hours and exposed to the aforementioned conditions for 48 hours and then counted using a hemocytometer ( n = 3 wells / group).
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