Fig. 2: ECCA induces cell apoptosis in a time- and dose-dependent manner.

A UACC62 cells were treated with ECCA at 0, 0.5, 1, and 5 µM, and at 12 h cells were collected and analyzed for cell apoptosis by FACS analysis. B Quantification of the percentage of the apoptotic cells in A. C Mel-Juso cells were treated with ECCA at 0 or 10 µM and at 12 h, cells were collected and analyzed for cell apoptosis by FACS analysis. D Quantification of the percentage of the apoptotic cells in C. E UACC62 cells treated with or without various concentrations of ECCA were lysed at different time points as indicated and immunoblotting analysis was performed for total and cleaved forms of Caspase8, -9, -3, and PARP. GAPDH as a housekeeping gene was used as a loading control. Red, green, blue, and black arrows, respectively, indicate the upregulated expression of cleaved (c) forms for PARP (c-PARP), Caspase8 (c-Caspase8), Caspase9 (c-Caspase9), and Caspase3 (c-Caspase3). F Quantification of the relative levels of c-Caspase8, c-Caspase9, c-Caspase3, and c-PARP in E, as relative fold change to the respective control cells, indicated by a dashed line as 1, after each phosphorylated protein was normalized to the corresponding total protein band. G UACC62 cells were treated with different conditions: DMSO (control), 10 µM z-VAD-FMK, 1 µM ECCA, z-VAD-FMK (10 µM) + ECCA (1 µM), 5 µM ECCA, z-VAD-FMK (10 µM) + ECCA (5 µM), and after 12 h, cells were collected and analyzed for cell apoptosis by FAC analysis. H Quantification of apoptotic cell percentage in G. All experiments were carried out three times, and error bars represent means + SD; P values are indicated with “*”, * indicates P < 0.05, ** indicates P < 0.01 when comparing ECCA-treated cells with the control group in B, D, F, and H by Student’s t test; Lines in H indicate comparisons of two specific groups.