Click on image to view larger version.
Figure 4. The effects of the inhibition of MAPK/ERK and PI3K pathways in human pulp cells. (a) Total extracts derived from human primary pulp cells treated with 1 mmol/L TEGDMA or 3 mmol/L TEGDMA for 15 min were separated on 10% SDS-PAGE, and immunoblotted with anti-P-Akt (ser 473) or anti-P-ERK 1/2. The amounts of total protein present in the extracts were determined by immunoblotting with anti-Akt, anti-tubulin, and anti-ERK 2. Experiments were performed 3 times, and a representative result is shown. (b) The ratio between P-Akt and total Akt in pulp cells after treatment with TEGDMA determined by densitometry. Asterisk indicates significant differences from untreated cell cultures (n = 3). (c) Induction of apoptosis and necrosis in human pulp cells. Cells were pre-treated with 50 µmol/L LY294002 and 40 µmol/L PD98056 for 30 min, and then further exposed to 1 mmol/L TEGDMA for 24 hrs. Apoptotic (annexin V+) and necrotic (PI+) cell populations were analyzed by flow cytometry as described. Viability of a cell population is expressed as the difference between the total cell population and the populations which stained with annexin V-FITC and PI. Values (means ± SD) from at least 4 independent experiments in duplicate are presented (n = 4); * indicates significant (p 
0.05), and ** indicates highly significant (p 0.01) differences from untreated control cultures (Mann-Whitney U-test). (d) Inhibition of Akt phosphorylation in human pulp cells. The cells were treated with 40 mmol/L LY294002 for 30 min, and then exposed to 1 mmol/L TEGDMA for 15 min. Cell lysates were separated on SDS-PAGE and blotted with anti-P-Akt or total Akt antibodies.
