Chios Mastiha Leaf Fractions Effects

Cells were grown in 6-well plates for 48 h in hormone-depleted medium and incubated for an additional 1 or 48–72 h with 20–100 μg/mL of Chios mastiha tree polar and medium-polar leaves fractions and/or 10 nM DEX, as indicated. Cells were washed in PBS 1X, lysed in buffer A (20 mM Tris pH:7.5, 250 mM NaCl, 0.5% Triton, 3 mM EDTA) supplemented with cocktail protease inhibitors, DTT and PMSF. After Bradford protein determination, cell extracts were electrophoresed in discontinuous SDS-PAGE and Western blotting with specific antibodies as previously described [48 (link)]. Enhanced chemiluminescence was used for the detection of the protein bands. β-actin and GAPDH expression levels were evaluated for the normalization of the GR, PEPCK, GS, procaspase-3, procaspase-9, Bcl-2, AMPK, and phosphο-AMPKα (Thr172) expression levels. In the case of MG-132 treatment, an inhibitor of the proteasome, HEK293 cells were pre-treated with 5μΜ MG-132, or DMSO, for 1 h [13 (link),14 (link)]. Then, the cell culture medium was replaced and HEK293 cells were further treated with 50 μg/mL leaves fractions or 10 nM DEX or DMSO, for 24 h. Cells were collected, lysed, and subjected to electrophoresis and Western blot analysis.