orescence and PI fluorescence gave different cell populations, where FITC and PI were designated 20142041 as viable cells, FITC and PI as apoptotic cells, and FITC and PI as late apoptotic or necrotic cells. Anthocyanins Inhibit HER2+ Breast Cancer Cells Caspase 3/7 activity assay Cells were grown to 7080% confluence, harvested and aliquoted into 96-well plates. Different doses of compounds were added to the plates the next day. Plates were incubated for an additional 48 h at 37uC. Aliquots of Alamar-Blue reagent were added directly to each well, the plates were incubated at 37uC for 3 h and the fluorescent signal was measured with an excitation at 530 nm and emission at 590 nm on ZS-2 plate reader. Then equal volume of caspase 3/7 activity assay reagent was added to each well and the luminescence signal was measured on ZS-2 plate reader. Data were normalized as luminescence relative to fluorescence. In vivo efficacy in xenograft models In vivo experiments were carried out under pathogen-free conditions at the animal facility in accordance with the institutional guidelines of the Chengdu Medical College Institutional Animal Care and Use Committee. All protocols were reviewed and approved by IACUC and HER2-positive breast cancer cell line MDA-MB-453 cells were resuspended to 26106 cells/100 ml in PBS and implanted subcutaneously into the flank region of 67-week-old female nude mice weighing 18 to 22 gram. When tumors reached 50 to 60 mm3 in volume, animals were randomly assigned to 3 groups, receiving either saline or peonidin-3-glucoside and cyaniding-3glucoside as oral gavage 7 times a week for a total of 25 days. Tumors were measured every 5 days with a caliper and tumor volume was calculated using the following formula: V = 4/3p3, where V = volume, w = width, l = length. Once the control tumors reached 1000 mm3, the animals were euthanized due to ethical requirements. After 25 days treatment, all animals were euthanized using overdosed CO2, and the tumor tissues were extracted for immunostaining and weighing. All values are expressed as the mean 6 SEM. Hit compounds inhibit the growth of HER2-postive cancer cell lines Third step, hit drugs that only inhibit HER2-positive cell line proliferation were chosen for a quantitative screening to generate IC50 values as described in the Materials and Methods section. The eight candidates’s structure were summarized in Hit compounds inhibit HER2 in HER2-postive cancer cell lines We first determined the phosphorylation status of the HER2 protein and its downstream mediator AKT to further confirm the anti-proliferation activities of the hit drugs are due to selective inhibition of HER2 protein. To evaluate the response of the cell lines to hit drugs, BT474, MDA-MB-453 and HCC1569 cells were treated with drugs for 6 h. Western blotting show that both peonidin-3-glucoside and cyanidin-3-glucoside significantly Halofuginone site reduce the phospho-HER2, phospho-AKTs, and phopspho-p44/42MAPK levels compared to control cells. Statistical analysis 11336787 In vitro data were reported as mean 6 SD, each treatment performed in duplicate or triplicate. Data were log-transformed to stabilize variances for proliferation assays. In vivo data were reported as mean 6 SEM. Values were analyzed using the Student’s t test or with one-way ANOVA when three groups were present. Statistical significance was considered as p0.05. Hit compounds induce apoptosis in HER2-postive cancer cell lines To further 
confirm the hit compounds activity, we performed
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