Fig. 3

Quince peel polyphenolic extract induces cell cycle arrest, caspase-independent apoptosis and ROS generation in LS174 cells. a Histogram analysis of the cell cycle phases in mock- and treated-LS174 cells with 5 µg/ml of Peph for 24 and 72 h by flow cytometry using propidium iodide assay. b Quince peel polyphenolic extract down-regulates the expression level of cyclin D1 protein. Whole cell lysates (30 µg), from LS174 cells treated with Peph (5 µg/ml) or vehicle for 24–72 h, were resolved on SDS-PAGE gel and probed with the indicated antibody for western blot analysis. β-actin was used as a control for equal loading. c Flow cytometry analysis using annexin V/7-AAD staining of Z-VAD-FMK (20 µM)-pretreated LS174 cells cultured in the absence (control) and presence of 5 µg/ml of Peph extract for 24 and 72 h. Annexin V/7AAD double staining discriminates the live cells (Annexin V−/7AAD−; bottom left quadrant), early apoptotic cells (Annexin V+/7AAD−; bottom right quadrant), late apoptotic or necrotic cells (Annexin V+/7AAD+; upper right quadrant), and dead cells (Annexin V−/7AAD+; upper left quadrant). d Compared to the positive control (Staurosporine), western blot analysis showed the absence of caspase 3, 9 activities in LS174 cells after incubation for 72 h with Peph extract. e Induction of caspase-independent pathways mediated by the increase of apoptosis-inducing factor AIF proteins after treatment by quince Peph extract. f ROS production was measured with CMH2DCFDA staining after 24 and 72 h of treatment with 5 µg/ml of peel polyphenolic extract. Fluorescence was used to detect ROS as described in “Methods”. The data are representative of three independent experiments. Data are reported as the mean ± SE of three separate experiments (*p < 0.05)