Annexin V assay.

<p>Apoptotic effect of flavone A at a concentration of 40 μM, on the more differentiated pancreatic Panc28 and colon CaCo 2 cancer cells (Fig 1A and 1B), as determined by Annexin V assay (green channel) six hours after treatment. Dapi (blue channel) is used to locate the nuclei of the cells. Cells treated with vehicle only (DMSO at a final concentration of 0.27%) served as a control. Activation of apoptosis on the poorly differentiated pancreatic MIA PaCa and colon HCT116 cancer cells (Figs 1C and 1D) by flavone B at a concentration of 40 μM, as determined by Annexin V assay (green channel) six hours after treatment. Control conditions are the same as described above and Dapi was used to locate nuclei.</p

Comparison of the effect of flavone A and flavone B on proliferative, and survival pathways.

<p>A and B: Detection of the activated and unphosphorylated forms of ERK, c-JUN, S6, AKT by immunoblot of total SDS extracts. Better differentiated Panc28 and CaCo 2 cells were treated with 40μM of flavone A (+A), and poorly differentiated MIA PaCa and HCT116 cells with flavone B (+B), or DMSO (-) the dissolution vehicle. After lysis and SDS-PAGE, membranes were probed with the indicated antibody. The membranes were reprobed for actin as a loading control, and a representative image is provided. The results shown are representative of three independent experiments. C and D: For quantification (graphs) the band densities from the treated/untreated conditions identified by (+) or (-), were normalized and calculated as percentages of the value for the untreated cells (100%), and shown averages ± standard deviations from three independent experiments (*p<0.05). E and F: Detection of phosphorylated ERK after treatment of CaCo 2 cells with flavone A and HCT116 cells with flavone B by immunofluorescence.</p

Analysis of downstream effector BAD after treatment with flavone A and flavone B.

<p>A and B: Detection of the loss of phosphorylation of BAD by immunoblot of total SDS extracts. Better differentiated Panc 28 and CaCo 2 cells were treated with 40μM of flavone A (+A), and poorly differentiated MIA PaCa and HCT116 cells with flavone B (+B), or DMSO (-) the dissolution vehicle. After lysis and SDS-PAGE, membranes were probed with an antibody specific to BAD phosphorylated at serine 112 or the unphosphorylated protein. The membranes were reprobed for actin as a loading control. The results shown are representative of three independent experiments. C and D: For quantification (graphs) the band densities from the treated/untreated conditions identified by (+) or (-), were normalized and calculated as percentages of the value for the untreated cells (100%), and shown averages ± standard deviations from three independent experiments (*p<0.05). E and F: Detection of phosphorylated BAD at serine 112 (red channel), after treatment of Panc 28 cells with flavone A and MIA PaCa cells with flavone B by immunofluorescence. Dapi (blue channel) was used to locate the nuclei.</p

Annexin V-FITC and propidium iodide flow cytometry.

<p>A. Apoptosis was detected using Annexin V-FITC and propidium iodide in Panc 28 cells treated with 40 μM flavone A, 9 hours after treatment. B. Detection of apoptosis in HCT 116 cells treated with 40 μM flavone B, 9 hours after treatment. C. Bar graph representation of apoptosis in Panc 28 and HCT 116 cells treated with flavone A and B respectively. D-E. Cell cycle determination using propidium iodide in Panc 28 cells treated 40 μM flavone A. F-G. Cell cycle determination in HCT 116 cells treated with 40 μM flavone B.</p

Analysis of c-JUN phosphorylation of threonines 91/93 and caspase 8 after treatment with 40 μM flavone B.

<p>A and B. Immunofluorescence of treated cells show expression of phospho-c-JUN (T91/T93) in SKBR3 cells (green channel) but not on HCT116 cells. Dapi (blue channel) was used to localize the nuclei. C. Immunoblot of phospho-c-JUN (T91/T93) using SDS extracts of poorly differentiated HCT116 cells treated with 40μM of flavone B (+B), or the dissolution vehicle DMSO (-). SDS lysates from SKBR3 cells treated with 10μM tamoxifen (+) were used as a positive control. After SDS-PAGE, nitrocellulose membranes were probed with an antibody specific to this phosphorylated form. D. Detection of caspase 8 by immunoblot of SDS lysates of HCT116 cells 1.5, 3, 6, 9 and 12 hours (lanes 2–6) after treatment with flavone B or vehicle (DMSO) for the control (C, lane 1) and SDS-PAGE. The membranes were probed with an antibody capable of detecting both the procaspase (54/55 kDa) and the fragments resultant after activation (43 and 18 kDa). The membranes were reprobed for actin or tubulin as a loading control. The results shown are representative of three independent experiments.</p

Analysis of caspase 9 after treatment with flavone A.

<p>Detection of activated caspase 9 by immunoblot of SDS extracts of A. CaCo 2 and B. Panc 28 cells 1.5, 3, 6, 9 and 12 hours (lanes 2–6) after treatment with flavone A or vehicle (DMSO) for the control (C, lane 1) and SDS-PAGE. The membranes were probed with an antibody capable of detecting both the procaspase (47 kDa) and the large fragments resultant after activation (37 and 35 kDa). The membranes were reprobed for actin or tubulin as a loading control. The results shown are representative of three independent experiments. The membranes were reprobed for actin or tubulin as a loading control. The results shown are representative of three independent experiments.</p