Sb202190

LPS was purchased from Sigma-Aldrich. Recombinant human VEGF, IL-1β and TNF-α were purchased from Cell Signaling Technology. Rabbit monoclonal antibodies against VE-cadherin, phospho-p38 (Thr180/Tyr182), p38, phospho-Src, Src, phospho-ERK1/2, ERK1/2, ICAM-1 and VCAM-1 were purchased from Cell Signaling Technology. Inhibitor of p38, SB202190, was purchased from Cell Signaling Technology. Rabbit polyclonal antibodies against 5-MTP was purchased from Abcam. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG were purchased from Santa Cruz.

To investigate the effects of the inhibitors on cell differentiation, cells were seeded at 2 × 10⁴ cells/well in a 1 M RGD-3-coated 12-well microplate. To check the effects of the inhibitor on cell mineralization, cells were seeded at 2 × 10⁴ cells/well in a 1 M RGD-3-coated 6-well microplate. Cells were challenged with 20 μM of one of three MAPK pathway inhibitors on day 6 in serum-free DMEM for 2 h, SP600125 (Cell Signaling Technology, Danvers, MA, USA) for c-Jun N-terminal kinase (JNK), SB202190 (Cell Signaling Technology) for p38 and PD98059 (Cell Signaling Technology) for extracellular signal-regulated kinase (ERK), respectively. Then, the medium was changed to DMEM supplemented with 10% FBS, 50 units/mL penicillin and 50 μg/mL streptomycin, and 10 mM β-GP and 50 μg/mL AA were added on day 7 when the cells reached confluence. ALP activity assays were performed on days 8 and 22. ARS staining was performed on day 31.

The non-tumorigenic immortalized human keratinocyte HaCaT cell line was obtained from Dr. Nancy Bigley (Wright State University), while the tumorigenic H-Ras transformed HaCaT II-4 cells were obtained from by Dr. Nancy Colburn (National Cancer Institute). The SCC cell line A431 was obtained from ATCC (Manassas, VA, USA). The three cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 8% fetal bovine serum and 250 U penicillin and 250 μg streptomycin. Neonatal human epidermal keratinocytes were maintained in KGM-Gold media as per manufacturer's instructions (Lonza, Walkersville, MD, USA). The Akt inhibitor MK2206 and the p38 inhibitor BIRB-796 were purchased from Selleckchem (Houston, TX, USA). The p38 inhibitor SB202190 was purchased from Cell Signaling Technology (Danvers, MA, USA). VD₃ was maintained as a 100 μM a stock in 100% ethanol (Cat # 17936, Sigma-Aldrich, St. Louis, MO, USA). VD₃ treatment were carried out in Dulbecco's modified Eagle's medium supplemented with 8% Charcoal-striped fetal bovine serum and 250 U penicillin and 250 μg streptomycin with a final EtOH concentration of 0.01%.

Crizotinib (PF-02341066) was obtained from ShangHai Biochempartner; cabozantinib (XL184), ceritinib (LDK378), lorlatinib (PF-06463922), trametinib (GSK1120212) from ActiveBiochem; foretinib from AdooQ Bioscience; brigatinib from Sellek; radicicol and rotenone from Cayman Chemical; 17-AAG (17-N-Allylamino-17-demethoxygeldanamycin) and GDC-0941 from LC Laboratories; TAE684 from ChemieTek; SB202190 from Cell Signaling Technology; SB239063 from Sigma; and cycloheximide and doxycycline hyclate from Nacalai Tesque. Each compound was prepared in dimethyl sulfoxide (DMSO), ethanol (WAKO) or distilled water for cell culture experiments.

In 24-well plates, 1–2 × 10⁴ LLC-PK1 cells/well were treated with 1, 10 or 100 nM CTS for 24, 48, and 72 h. In order to investigate the involvement of intracellular signaling pathways, 100 nM CTS were incubated with or without inhibitors of Src (SU6656, 10 μM; Sigma-Aldrich, St. Louis, MO, USA), ERK1/2-MEK1/2 (U0126, 10 μM; Cell Signaling Technology, Danvers, MA, USA), PI3K (LY294002, 5 μM; Cell Signaling Technology, Danvers, MA, USA), p38 (SB202190, 10 μM; Cell Signaling Technology, Danvers, MA, USA), JNK1/2 (SP600125, 1.5 μM; Cell Signaling Technology, Danvers, MA, USA), or GSK-3β (BIO, 0.5, 1, and 5 μM; Sigma-Aldrich, St. Louis, MO, USA) for 72 h. For MβCD (Sigma-Aldrich, St. Louis, MO, USA) assay, cells were pretreated with 10 mM MβCD for 30 min and then 1 mM MβCD + 100 nM TCB in 2.5% FBS for 72 h. At these time points, two wells from each group were trypsinized and the number of Trypan blue-viable cells was counted in Neubauer chamber (hemocytometer).

The IPEC-J2 intestinal porcine enterocyte cell line (DSMZ, Braunschweig, Germany) was maintained, as previously described [11 (link)]. Moreover, the cells were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium supplemented with 5% fetal bovine serum, 1% insulin–transferrin–selenium-X, and 1% (v/v) penicillin–streptomycin [55 (link)]. The cells were maintained at 37 °C in a humidified atmosphere of 5% CO₂. The IPEC-J2 cells were incubated with different treatments, including recombinant HBEGF (Mybiosource, San Diego, CA, USA), LY2944002 (Cell Signaling Technology, Danvers, MA, USA), SB202190 (Cell Signaling Technology, USA), and the AMPK inhibitor (dorsomorphin dihydrochloride; Santa Cruz Biotechnology, Dallas, TX, USA) at different intervals, as indicated in the figure legends.