Sb203580

Camptothecin (CPT) was purchased from Sigma-Aldrich, the p38 inhibitors SB202190 from SA Biosciences, Frederick, MD, USA and SB203580 from BioSource, Nivelles, Belgium.

When needed, samples were treated with 10 µM of MAPK-p38 specific inhibitor, SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole] (BioSource, LabClinics, Barcelona, Spain). Also, 20 µM of SP600125 [Anthra(1,9-cd)pyrazol-6(2H)-one] (Biosource, LabClinics), a pharmacological inhibitor of c-Jun NH2-terminal kinase (JNK), was used. These concentrations have been proven effective in a number of studies [78 (link),79 (link)]. The inhibitors were dissolved in dimethyl sulfoxide (DMSO; Sigma, Madrid, Spain). An equal dose of DMSO (vehicle) was added to the control dishes. The final DMSO concentration in the media was always ≤0.01%, which was proven not to influence the cell growth rate in the cultures (data not shown). On the third day after seeding (long-term proliferation assays) or on the fourth day after seeding (short-term assays), the medium was renewed and supplemented with the inhibitor or the vehicle 1 h prior to the MF- or sham-exposure onset.

All-trans retinoic acid (ATRA), cytosine-arabinofuranoside (AraC), 2′7′-dichloro-fluorescein diacetate (DCF-DA), 3-(4,5-dimethylthiazol-2yl)2,5-dyphenyl-2H-tetrazolium bromide (MTT), hydrogen peroxide solution and quercetin dihydrate were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Culture medium (Dulbecco’ Modified Eagle’s Medium (DMEM)), foetal bovine serum (FBS), trypsin, antibiotics, L-glutamine and supplements for differentiation of P19 cells were also purchased from Sigma-Aldrich (St. Louis, MO, USA) or Gibco (Paisley, UK). SP600125 and SB203580 were from Alfa Aesar (Ward Hill, MA, USA). EEP was obtained as previously described [57 (link)]. All other chemical used were of analytical grade.
In order to confirm the authenticity of the propolis, infrared spectra of EEP were recorded with an ABB Bomem Mb102 Fourier transform infrared (FTIR) spectrometer at room temperature. For each infrared spectrum, 30 interferograms were collected with a nominal resolution of 4 cm⁻¹. The reference spectra were recorded under the same conditions. The spectra were collected on a MKII Golden Gate single reflection attenuated total reflection (ATR) accessory.

We purchased sheep anti-rabbit antibodies of CD29, CD34, CD44, CD45, and fluorescein isothiocyanate- (FITC-) labeled IgG secondary antibody from eBioscience (San Diego, CA). Sheep polyclonal anti-human antibodies to alpha-fetoprotein (AFP), albumin (ALB) and β-actin, and FITC-labeled IgG secondary antibody were obtained from Sigma (St. Louis, MO). Antibodies to DNMT1 and phosphorylated p38 were obtained from Cell Signaling Technology (Beverly, MA). HGF was purchased from Proteintech (Rocky Hill, NJ). Zebularine was purchased from Berry and Associates (Dexter, MI).
The p38 inhibitor SB203580 was obtained from Merck (Germany). A stock solution of SB203580 was prepared in dimethyl sulfoxide (DMSO). SB203580 was diluted in Dulbecco modified Eagle medium (DMEM; Gibco, Rockville, MD) and was added to cell culture 1 h before stimulation with HGF and kept further during the exposure to this compound. A stock solution of zebularine for cellular assays was prepared in DMSO and then diluted in the optimal medium to the final concentrations. Indocyanine green (ICG) was obtained from Aladdin (Shanghai, China).

To examine the signaling pathways involved in cytokine secretion, decidualized T-HESC cells were pretreated with 10µM SB203580 (p38 MAPK inhibitor, Gibco), 10 µM SP600125 (Jnk1/2 inhibitor, Sigma), 2.5 µM BAY11-7082 (NF-κB inhibitor, Sigma) or vehicle (DMSO). These reagents were added one hour before infection with B. abortus and were kept throughout the experiment (48 h). Cell viability after incubation with these inhibitors was higher than 90%, as assessed by staining with trypan blue.