Jnk in 8

To elucidate the roles of signal transduction proteins to regulate splicing changes, each protein known to be regulated downstream of TCR signaling was targeted by at least two different inhibitors. Human primary CD4+ CD45RO- T cells were incubated with individual inhibitors for 1 hr and stimulated with bound anti-CD3 and soluble anti-CD28 antibodies for 48 hr. Cells were then harvested for RNA purification and low-cycle RT-PCR analysis for splicing quantifications. The inhibitors utilized are as follows: PCKi: R0-31-8220 (Selleckchem: S7207) and Go6983 (Selleckchem: S2911), p38i: SB20350 (Selleckchem: S1076) and Skepinone-L (Selleckchem: S7214), NFATi: Cyclosporin A (Selleckchem: S2286) and FK506 (Selleckchem: S5003), AKTi: MK-2206 (Selleckchem:S1078) and Ipatasertib (Selleckchem:S2808), JNKi: SP600215 (Selleckchem: S1460) and JNK-IN-8 (Selleckchem: S4901) and Tanzisertib (Selleckchem: S8490).

Recombinant human matrix metalloproteinases‐7 protein was obtained from AmyJet Scientific. The chemical inhibitors for the MAPK pathway (ie U0126, SB203580 and JNK‐IN‐8) were purchased from Selleck Chemicals. Antibodies (Abs) against phospho‐ERK, phospho‐JNK, phospho‐p38, phospho‐β‐catenin, phospho‐AKT, phospho‐mTOR, phospho‐p65, ERK, JNK, p38, β‐catenin, AKT, PI3K p110, PI3K p85 and mTOR, p65 were obtained from Cell Signaling Technology. Anti‐AQP‐1(AF5231) and Anti‐MMP‐7(AF0218) were brought from Affinity Biosciences LTD. HRP‐conjugated Goat Anti‐Rabbit IgG and HRP‐conjugated Goat Anti‐Mouse IgG were bought from Proteintech.

Human phospho-kinase array kits (ARY003B, R&D Systems, Minneapolis, MN, USA) were blotted as per manufacturer's protocol. HCC38 cells were sorted as previously described [7 (link)]. Whole cell lysates were prepared with RIPA lysis buffer (100 mM Tris pH 7.5, 150 mM sodium chloride, 0.1% deoxycholate, 0.1% SDS, 50 mM NaF, Protease inhibitor cocktail (Roche), 2 mM PMSF, 2 mM sodium orthovanadate) combined with scraping and the lysates cleared by centrifugation. Standard Western blotting procedures were performed. The following primary antibodies were used for immunoblotting at a dilution of 1:1000: AXL (8661, Cell Signaling Technology, Danvers, MA, USA), c-Jun (2315, Cell Signaling Technology), pS63 c-Jun (91,952, Cell Signaling Technology), Hsp90 (sc-13119, Santa Cruz, Dallas, TX, USA), or 1:4000 β-actin (MABT825, MilliporeSigma). Treatments with 5 μM JNK–IN–8 (Selleckchem) or vehicle control (DMSO) were performed for 24 h prior to harvesting lysates. All blots or gels derive from the same experiment and were processed in parallel. See Supplementary Material for unedited blots (Supplementary Figs. S5 and S6).

Jun N-terminal kinase inhibitor II SP600125 (JNKi) was obtained from Calbiochem, JNK-IN-8 from Selleckchem, recombinant human TRAIL (rhTRAIL) from R&D Systems, and Gemcitabine from Elly Lilly. Products were reconstituted as recommended by the manufacturer.
The following antibodies were used: phospho-c-Jun (Cell Signaling Technologies), CD133-APC (Miltenyi Biotech), SSEA1-FITC (Santa Cruz Biotechnology, Inc.), DR4 antibody-PE Mouse IgG1B, DR5 antibody-FITC Mouse IgG2B and DcR1 antibody-APC Mouse IgG1 (R&D Systems).
Small interfering RNA for SAPK/JNK (#6232) and control (#6568) was obtained from SignalSilence ^® (Cell Signaling Technologies).

Dehydrocrenatidine (≥98% purity) was purchased from ChemFaces (CheCheng Rd, Wuhan, PRC). The stock solution (100 mM) was prepared by dissolving dehydrocrenatidine in dimethyl sulfoxide (DMSO) and kept at −20 °C for further use. The JNK-specific inhibitor (SP600125) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and a stock solution of 20 mM was prepared using DMSO. Another JNK-specific inhibitor (JNK-in-8) was purchased from Selleck Chemicals (Houston, TX, USA), and a stock solution of 5 µM was prepared using DMSO. The primary and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The phenylindole (DAPI) dye and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St Louis, MO, USA), and stock solutions were prepared at a concentration of 10 mg/mL and 5mg/mL, respectively.

The following chemical inhibitors were dissolved in DMSO (Sigma) (unless stated otherwise) as stock solutions and then subsequently diluted in culture media: Vorinostat/SAHA (Biotang), LBH-589 (Biotang), MS-275 (Biotang), SP600125 (LC Lab), JNK-IN-8 (Selleck Chem), JNK-IN-9 (Selleck Chem), and SR3576 (ApexBio).

Pirfenidone (10 to 300 μg/ml [71 (link), 116 –118 (link)]) was obtained from Chemietek. AraC (1 to 10 μM [119 (link), 120 ]), LY2228820 (0.03 to 2 μM [68 (link), 69 (link), 121 (link), 122 (link)]), BIRB796 (BIRB, 5 μM [70 (link), 123 (link)–126 (link)]), and JNK-IN-8 (1 μM [127 (link)] were from Selleckchem. KU55933 (10 μM [128 (link)], tested but was toxic for the cells tested), BAY 11-7082 (10 μM [78 (link)]), and D-luciferin were from Cayman Chemical and doxorubicin (10 to 500 nM [129 (link)]) was from Tocris Bioscience.