Pd98059

Selumetinib (AZD6244, MEK inhibitor), trametinib (GSK1120212, MEK inhibitor), KX2-391 (Src inhibitor) and tofacitinib (CP-690550, JAK3 inhibitor) were purchased from Selleck Chemicals (Houston, TX, USA). PD98059 (MEK inhibitor) and ruxolitinib (INCB081424, JAK1/2 inhibitor) were purchased from LC Laboratories (Woburn, MA, USA). LY5 (STAT3 inhibitor [67 (link)]) was synthesized by Chenglong Li's Lab (College of Pharmacy, The Ohio State University). The drug compounds were dissolved in sterile dimethyl sulfoxide (DMSO) to make a 20 mM stock solution stored at −20°C.

Seven human pancreatic cancer cell lines, HPAF-II, BxPC3, Capan2, CFPAC-1, HPAC, SU.86.86, and MIA PaCa-2 cells were obtained from American Type Culture Collection (ATCC). These cell lines are cultured in complete growth medium as the previous study^{11 (link)}. HPAF-II was maintained in MEM. BxPC3 and SU.86.86 were maintained in RPMI-1640. Capan2 was maintained in MyCoy’s 5A. CFPAC-1 was maintained in IMDM. HPAC was maintained in DMEN/F12. All the media were supplied with 10% FBS. MIA PaCa-2 was maintained in DMEM with 5% horse serum. And the cells were incubated at 37 °C with 5% CO₂ supplement. All medium supplements were purchased from ThermoFisher (Waltham, MA, US). MEK kinase inhibitor U0126 and PD98059 were purchased from LC Laboratories (Woburn, MA, US). MUC1 inhibitor Go-201 and NF-κB inhibitor BAY11-7082 were purchased from Sigma-Aldrich (St. Louis, MI, US).

Isolated mouse BMCs were suspended in StemSpan serum-free medium (StemCell Technologies, Vancouver, BC, Canada) containing 20% mouse plasma and then plated into 24-well tissue culture plates with 500 µl of cell suspension (containing 5 × 10⁶ cells) per well. Culture of cells was conducted without or with lipopolysaccharide (LPS, E. coli 0111:B4, 20 ng/ml, Sigma-Aldrich Co., LLC, St. Louis, MO, USA) stimulation in the absence and presence of specific mitogen-activated protein kinase kinase1/2 (MEK1/2) inhibitor PD98059 (25 µM, LC Laboratories, Woburn, MA, USA) for 18 h.

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), phosphate buffer saline (PBS), Horse serum (HS), antibiotics and antimycotics, trypsin 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Hoechst 33342 were purchased from Invitrogen (Carlsbad, CA, USA). PD98059, SB415286, and LY294002 were obtained from LC laboratories (Woburn, MA, USA). ECL plus kit were purchased from Amersham Bioscience (Aylesbury, UK). 2′, 7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), ethyl alcohol (absolute ethanol), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Cell lysis buffer, antibodies against phospho-MEK 1/2, phospho-ERK 1/2, ERK, phospho-PI3K, phospho-Akt, Akt, phospho-GSK3β, GSK3β were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibody against β-actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals used were of analytical grade.

HBEC were obtained from Clonetics (San Diego, CA, USA) and cultured in BEG media (Lonza, Walkersville, USA). Human lung carcinoma cell lines, including small-cell lung cancer cell (COLO677, COLO668, H82, DMS79, H526, SHP77 and H123) and NSCLC cell lines (ADC: H1299, H2030, H23, A549, H322, H1650, H1975, D51, D54, H125, D117, D97 and BEN/97 as well as SCC: H2170, H226, H157), were purchased from the American Type Culture Collection (ATCC, Rockville, USA) or established in our laboratory (D51, D54, D117 and D97) [8 (link)]. Cells were grown in RPMI 1640 medium (Biochrom AG, Berlin, Germany) or Leibovitz 15 media (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Biochrom AG) and maintained in a humidified atmosphere with 5% CO2 at 37°C.
For demethylation, 5 μM of DAC (Sigma Chemical Co., St Louis, MO USA) was added into the medium on days 0, 2 and harvested in days 4 when cells around 60 % confluence. For deacetylation tests, cells were treated with 1 μM of TSA (Sigma) for 1 day.
To study the effect of TGF-β1 on EMT, cells were starved in serum-free medium overnight and treated with 5 ng/ml of human recombinant TGF-β1 (PeproTech, New Jersey, USA) and/or 10 μM of PD98059 (LC laboratories, Woburn, MA USA) for 24 h.