AE treatment increases beclin1 and LC3B-II expression in OVCAR3 and SW626 cells.

<p>Immunostaining of beclin1 and LC3B-II in OVCAR3 and SW626 control cells and after being treated with 300 µg/ml AE (A, B, E and F). The histograms show the percentage of beclin1 (C and D) and LC3B-II (G and H) immunopositive cells as a percentage of total cells. OVCAR3 and SW626 cells were cultured and grown for 2 days in DMEM in the presence of 10% serum as described under Materials and Methods. After this period, the cultures were fed with medium containing 10% serum and AE for 24 hours. Cells were photographed at 400 x magnification. Expression of beclin1 in total protein of OVCAR3 (I) and SW626 (J) cells and expression of LC3B-II in total protein of OVCAR3 (M) and SW626 (N) cells after being treated with AE (300 µg/ml) for 24 hrs. Representative photograph of western blot for beclin1 and LC3B-II are shown on the top. β-actin was detected as control for each blot. Mean <u>+</u> S.E.M. values of densitometric ratio of beclin1 (K and L) and LC3B-II (O and P) with β-actin are shown on the bottom of each respective gel. Q–T: Quercetin treatment (5 µg/ml for 48 hour) induces expression of beclin1 (Q, R) and LC3B-II (S, T) in OVCAR3 cells. After treatment, protein from OVCAR3 and SW626 cells was extracted and 50 µg proteins were electrophoresed on SDS-PAGE. The protein was then transferred to a nitrocellulose membrane and immunoblotted against beclin1 and LC3B-II antibodies. After immunodetection, beclin1 and LC3B-II positive bands were measured densitometrically and normalized with β-actin values. The values are means <u>+</u> S.E.M. of 6 independent experiments. *, p<0.05, as compared with control group. Bar=50 µm.