Jnk inhibitor sp 600125

We purchased JNK inhibitor SP600125 from Cayman Chemical (Ann Arbor, MI, USA), and transforming growth factor alpha (TGF‐α) from R&D Systems (Minneapolis, MN, USA). Primary antibodies used in immunohistochemical and immunofluorescence assays included rabbit anti‐phospho‐JNK antibody (1:50; Cell Signaling Technology, Danvers, MA, USA), rat anti‐mouse CD45 antibody (1:50; BD Biosciences, San Jose, CA, USA), rat anti‐mouse F4/80 antibody (1:100; eBioscience, San Diego, CA, USA), rabbit anti‐Ki‐67 antibody (1:100; Abcam, Cambridge, UK), mouse anti‐α‐smooth muscle actin (α‐SMA) antibody (1:50; Santa Cruz, Santa Cruz, CA, USA), biotin hamster anti‐mouse CD11c antibody (1:100; BD Biosciences), rabbit anti‐cleaved caspase3 antibody (1:1600; Cell Signaling Technology) and rabbit anti‐CD8 antibody (clone EP1150Y, 1:250; Epitomics, Burlingame, CA, USA). Primary antibodies used in immunoblotting analysis included rabbit anti‐phospho‐JNK antibody (1:1000; Cell Signaling Technology), rabbit anti‐JNK antibody (1:1000; Cell Signaling Technology), rabbit anti‐phospho‐Stat3 antibody (1:2000; Cell Signaling Technology) and mouse anti‐Stat3 antibody (1:1000; Cell Signaling Technology).

NMuMG cells were a kind gift from Rik Derynck (University of California, San Francisco) and maintained in DMEM+Glutamax supplemented with 10% fetal bovine serum (FBS), sodium pyruvate, nonessential amino acids (Thermo Scientific), bovine insulin solution (5 μg/ml; Sigma) and MycoZAP (Lonza). MycoZAP is included to prevent Mycoplasma contamination. Cells were maintained in a humidified atmosphere of 5% CO₂ at 37°C. For experiments, cells were plated at 1.6 × 10⁴ cells/cm² in 100-mm plates or six-well dishes for protein harvesting and eight-well chamber slides for imaging. Cells were incubated for 24 h before addition of media containing one of the following treatments: TGF-β1 (2 ng/ml; Humanzyme), the TβR1 blocker SB 431542 (SB; 5 µM; Sigma), Smad3 inhibitor SIS3 (10 µM; Selleckchem), p38 inhibitor SB 202190 (20 µM; Cayman), JNK inhibitor SP 600125 (20 µM; Cayman), ERK1/2 inhibitor SCH 772984 (0.1 µM; Cayman), or vehicle (veh; dimethyl sulfoxide [DMSO]). 293FT cells were obtained from Thermo Scientific and maintained in DMEM+Glutamax supplemented with 10% FBS, sodium pyruvate, nonessential amino acids (Thermo Scientific), and MycoZAP (Lonza).