Analyzer2200

Example 4

Cardiomyocytes were prepared as described above. Cells were co-treated with 1 μM of doxorubicin and either myricetin (FIG. 7A), myricetrin (FIG. 7B), or dihydromyricetin (FIG. 7C) for 3 days. Following treatment, the samples were incubated with a CellEvent dye to indicate apoptosis-positive cells, and a second dye to identify cell nuclei. Cells were imaged using the INCell Analyzer2200, and images were analyzed to quantify the percentage of apoptotic cells. Data are presented from two independent sets of screening where each data point was obtained from triplicate.

Cardiomyocytes co-treated with doxorubicin and either myricetin (FIG. 7A), myricitrin (FIG. 7B), or dihydromyricetin (FIG. 7C) exhibited protective effects against apoptosis, with half minimal inhibitory concentrations (IC50; e.g., the drug concentration that induces 50 percent apoptosis) of 20.46 μM, 38.48 μM, 40.48 μM, respectively.

Example 2

Human iPSC-derived cardiomyocytes were prepared by differentiating induced pluripotent stem cells into cardiomyocytes. Cells were cultured for 4 days post-differentiation, changing media at day 3, before performing experiments. Cardiomyocytes were treated with 1.25 μM doxorubicin (FIG. 4A), or co-treated with 1.25 μM of doxorubicin and 79 μM myricetin (FIG. 4B) for 2 days. Following treatment, the samples were incubated with a tetramethylrhodamine methyl ester (TMRM) dye to indicate mitochondrial health, and Hoechst 33342 to identify cell nuclei. Cells were imaged using the INCell Analyzer2200. Representative images are presented for each sample, wherein a decrease in TMRM signal indicates an increase in mitochondrial toxicity. Myricetin was a potent protector against doxorubicin-induced mitochondrial toxicity, as indicated by a greater TMRM signal in cells co-treated with 1.25 μM doxorubicin and 79 μM myricetin (FIG. 4B) as compared to cells treated with 1.25 μM doxorubicin in the absence of myricetin (FIG. 4A).

Example 19

Cell samples were prepared by differentiating induced pluripotent stem cells into cardiomyocytes. Cells were cultured for 4 days post-differentiation, changing media at day 3, before performing experiments. Samples were either mock-treated (FIG. 27A), treated with 1 μM of doxorubicin (FIG. 27B), co-treated with 1 μM of doxorubicin and 16 μM dexrazoxane (FIG. 27C), or co-treated with 1 μM of doxorubicin and 116 μM dexrazoxane (FIG. 27D) for 7 days. Following treatment, the samples were incubated with a tetramethylrhodamine methyl ester (TMRM) dye to indicate mitochondrial health. Cells were imaged using the INCell Analyzer2200, and images were analyzed by CellProfiler to quantify the percentage of TMRM-negative cells. Representative images are presented for each sample, wherein loss of TMRM signal represents mitochondrial toxicity.

Cardiomyocytes exposed to either doxorubicin (FIG. 27B) or co-treated with doxorubicin and dexrazoxane (FIG. 27C) exhibited an increase in mitochondrial toxicity as indicated by a noticeable decrease in TMRM-positive cells as compared to mock-treated cardiomyocytes (FIG. 27A). Treatment of cardiomyocytes with doxorubicin and vitexin (FIG. 27D) demonstrated improved long term mitochondrial protection, as compared to cardiomyocytes exposed to either doxorubicin (FIG. 27B) or doxorubicin and dexrazoxane (FIG. 27C).

Example 4

Cardiomyocytes were prepared as described above. Cells were co-treated with 1 μM of doxorubicin and either myricetin (FIG. 7A), myricetrin (FIG. 7B), or dihydromyricetin (FIG. 7C) for 3 days. Following treatment, the samples were incubated with a CellEvent dye to indicate apoptosis-positive cells, and a second dye to identify cell nuclei. Cells were imaged using the INCell Analyzer2200, and images were analyzed to quantify the percentage of apoptotic cells. Data are presented from two independent sets of screening where each data point was obtained from triplicate.

Cardiomyocytes co-treated with doxorubicin and either myricetin (FIG. 7A), myricitrin (FIG. 7B), or dihydromyricetin (FIG. 7C) exhibited protective effects against apoptosis, with half minimal inhibitory concentrations (IC50; e.g., the drug concentration that induces 50 percent apoptosis) of 20.46 μM, 38.48 μM, 40.48 μM, respectively.

Example 4

Cardiomyocytes were prepared as described above. Cells were co-treated with 1 μM of doxorubicin and either myricetin (FIG. 7A), myricetrin (FIG. 7B), or dihydromyricetin (FIG. 7C) for 3 days. Following treatment, the samples were incubated with a CellEvent dye to indicate apoptosis-positive cells, and a second dye to identify cell nuclei. Cells were imaged using the INCell Analyzer2200, and images were analyzed to quantify the percentage of apoptotic cells. Data are presented from two independent sets of screening where each data point was obtained from triplicate.

Cardiomyocytes co-treated with doxorubicin and either myricetin (FIG. 7A), myricitrin (FIG. 7B), or dihydromyricetin (FIG. 7C) exhibited protective effects against apoptosis, with half minimal inhibitory concentrations (IC50; e.g., the drug concentration that induces 50 percent apoptosis) of 20.46 μM, 38.48 μM, 40.48 μM, respectively.