The 254 PKIs that were tested in this study constitute part of the Chemical Validation Library (CVL), which is the core library of the Nested Chemical Library of Vichem and consists of launched kinase inhibitor drugs and compounds in clinical trials and in preclinical development (http://www.vichem.hu/nested_chemical_library.html ) [21] (link). Four of these 254 PKIs were further studied; PP2 is an inhibitor of Src family tyrosine kinases with higher potency for Lck and Fyn. Compound 5 also inhibits Src family kinases with Lck being the most sensitive. CI-1040 and PD 198306 are MEK inhibitors. PP2, CI-1040 and PD 198306 have already been used in pharmacological studies [22] (link)–[24] (link). Inhibitors were synthesized according to literature procedures: PP2 [25] (link), compound 5 [26] (link), CI-1040 and PD 198306 [27] (link). A-770041 (Axon Medchem) is a selective inhibitor of Lck, while U0126 (Gibco) is a selective inhibitor of MEK1 and MEK2. All inhibitors were dissolved in DMSO.
U0126
Manufactured by Thermo Fisher Scientific
Sourced in United States
U0126 is a specific inhibitor of the mitogen-activated protein kinase kinase (MEK1/2) enzymes. It blocks the activation of extracellular signal-regulated kinases (ERK1/2), thereby inhibiting the ERK signaling pathway.
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Lab products found in correlation
Sb203580, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
A770041, by Axon Medchem (1 mentions)
Smgm 2 medium, by Lonza (1 mentions)
Ang 2, by Thermo Fisher Scientific (1 mentions)
Angii, by Bachem (1 mentions)
Hpasmc, by ScienCell (1 mentions)
17 aag, by Thermo Fisher Scientific (1 mentions)
Su5402, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Igf 1, by Thermo Fisher Scientific (1 mentions)
Pd98059, by Thermo Fisher Scientific (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
17β estradiol, by Merck Group (1 mentions)
Rapamycin, by LC Laboratories (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Anti cd3, by BD (1 mentions)
Anti cd28, by BD (1 mentions)
Mc3t3 e1 cell, by American Type Culture Collection (1 mentions)
12 protocols using u0126
1
Kinase Inhibitor Compound Characterization
2
Signaling Pathway Inhibitors in Cell Experiments
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Specific inhibitors used in cell experiments are as follows: p38-specific inhibitor (SB203580, Gibco, USA), ERK-specific inhibitor (U0126, Gibco, USA), and NF-κB p65-specific inhibitor (BAY11-7082, Gibco, USA). The cells were pretreated with these pharmaceutical inhibitors for 1 hour, followed by other treatments.
3
IPAH Patient-Derived Smooth Muscle Cells
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Human PASMCs were provided as de-identified samples from a patient with idiopathic pulmonary arterial hypertension (IPAH), or a failed donor (FD) control, by the PHBI Research Network and maintained in Sm-GM2 medium (Lonza) at 37°C in a 5% CO2 humidified incubator. Some experiments employed human pulmonary artery smooth muscle cells (hPASMCs) from ScienCell (No. 3110), cultured in medium from the same company (No. 1101). Human PASMCs were used at less than passage 9 for all studies. Prior to treatment with ANG II (1 µM; Bachem), hPASMCs were cultured in medium containing 0.1% FBS for 18 h to induce growth arrest. Cells were pretreated with 5 µM PD98059 (LC Labs; P-4313) or 10 µM U0126 (Fisher; PR-V1121) for 30 min before ANG II addition. Studies using human PASMCs were deemed Institutional Review Board exempt.
4
Detailed Methodology for Pharmacological Agents
5
Pharmacological Modulation in Zebrafish
6
TGF-β1 and IGF-1 Regulate Collagen Expression
7
Pharmacological Agents for Myocardial Infarct
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17β-estradiol and chelerythrine chloride (CC) were from Sigma-Aldrich (St. Louis, MO). 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002) and U0126 were from Invitrogen. All primary antibodies were from Cell Signaling. Except for E2, which was diluted in ethanol (final ethanol concentration in perfusion buffer was <0.001%), all of the other pharmacological agents were dissolved in DMSO (final DMSO concentration in perfusion buffer was 0.01%). We previously showed that this concentration of DMSO does not affect heart function or myocardial infarct size after I/R [22 (link)]. We also did not present in this manuscript the control inhibitors groups because we already showed that at the concentrations used, all these inhibitors did not change the heart functional recovery and myocardial infarction as compared to normal controls [3 (link),22 (link)]
8
Jurkat and HEK293 Cell Culture Protocols
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The JSL1 clonal population of Jurkat cells was cultured in RPMI supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Lynch and Weiss 2000 (link)). HEK293 cells were cultured in DMEM with 5% heat-inactivated FBS. For stimulations, Jurkat T cells were cultured with 20 ng/mL PMA (Sigma-Aldrich) for 48 h (RT–PCR), 2 h (Western analysis of phosphoproteins), or the indicated time points. CD4+ peripheral blood T cells were obtained from the University of Pennsylvania Human Immunology Core (IRB protocol #811028) with an average purity of 90%–95%. CD4+ cells were maintained in RPMI supplemented with 10% heat-inactivated FBS for unstimulated conditions or were additionally stimulated with anti-CD3 (BD Biosciences, 555336) and anti-CD28 (BD Biosciences, 348040) for 48 h. For stimulations, plate wells were coated with anti-CD3 at 2.5 μg/mL, and additional soluble anti-CD3 and anti-CD28 were added to a final concentration of 2.5 μg/mL. For inhibition studies, cells were pretreated for 1 or 3 h (JNK-IN-8) with the following inhibitors before stimulating as described above: 50 μM SP600125 (Calbiochem, 420119), 3 μM JNK-IN-8 (Calbiochem, 420150), 1 μM triciribine (Calbiochem, 124012), 100 nM rapamycin (LC Laboratories, R-5000), and 20 μM U0126 (Invitrogen, PHZ1283). Transcription inhibition was achieved by incubation of cells with actinomycin D at a final concentration of 5 μg/mL.
9
Sema4D Regulates Osteoblast Differentiation
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MC3T3-E1 cells (ATCC, Manassas, VA, USA) were incubated in a 24-well plate (1 × 105 cells/well) and cultured in Basal Medium supplemented with 50 μg/mL ascorbic acid and 5 mM β-glycerophosphate in the presence or absence of anti-Sema4D mAb (50 µg/mL) or control IgG (50 µg/mL) and in the presence or absence of TPs or recombinant human Sema4D (Abcam, Waltham, MA, USA).
After 7 days, Alkaline Phosphatase (ALP) staining was performed with Alkaline Phosphatase (ALP) Staining kit (Sigma-Aldrich) following the manufacturer’s instruction. After 21 days, Alizarin Red S (Sigma-Aldrich) staining was performed. In another experiment, MC3T3-E1 cells were cultured with U0126 (ERK inhibitor; 10 μM, Thermo Fisher Scientific) or LY294002 (Akt inhibitor; 10 μM, Thermo Fisher Scientific). After 7 days of culture, the cells were stained with ALP Staining kit.
After 7 days, Alkaline Phosphatase (ALP) staining was performed with Alkaline Phosphatase (ALP) Staining kit (Sigma-Aldrich) following the manufacturer’s instruction. After 21 days, Alizarin Red S (Sigma-Aldrich) staining was performed. In another experiment, MC3T3-E1 cells were cultured with U0126 (ERK inhibitor; 10 μM, Thermo Fisher Scientific) or LY294002 (Akt inhibitor; 10 μM, Thermo Fisher Scientific). After 7 days of culture, the cells were stained with ALP Staining kit.
10
Alendronate Modulates Cell Signaling in Osteoblasts
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Nitrogen-containing BP, alendronate sodium salt trihydrate (FOS) was purchased from Wako in Japan. SCGMTM BulletKitTM, N-acetyl-cysteine (NAC), fluorescein isothiocyanate (FITC)-conjugated annexin V, and ECL chemiluminescence were acquired from Sigma in China. PTHrP, cell-permeable 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), U0126 (inhibitor of ERK1/2), SB203580 (inhibitor of p38), sanguinarine chloride (inhibitor of MKP1), lipofectamine LTX transfection reagent, Tris HCl, NaCl, EDTA, 1% Triton X-100, 1% sodium hydroxide, 0.1% sodium dodecyl sulfate (SDS), protease inhibitor cocktail, cocktail Set II, bovine serum albumin (BSA), Considering bicinchoninic acid (BCA), nitrocellulose membrane were obtained from ThermoFisher in China. In addition, α-Tubulin, peroxidase-conjugated goat anti-rabbit IgG and the antibodies of p-ERK1/2, ERK1/2, p-Akt, Akt, p-p38, p38, p-JNK, JNK, MKP-1, and p-p66Shc (Ser36) were acquired from ThermoFisher in China. Propidium iodide was purchased from Solarbio in China.
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