Figure 2.

J. rubens fractions cytotoxicity measured in a cancer cell line array. (A) The J. rubens chloroform:MeOH (2:1 v/v) Fall extract underwent VLC chromatographic separation over silica RP and eluted with increased MeOH percentage, resulting in fractions of different colors. (B) A549 cells were treated with J. rubens Fall extract and every elution fraction at 100 µg/ml for 48 h (n = 2). Cell death is presented as mean ± SE. Statistical significance was analyzed with Wilcoxon test (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Elutions 60 to 80% MeOH were pulled together and the resulting VLC80 was screened for its cytotoxic effect on A549, NCI-H460, PC3, lnCAP, HT29, SW480, MCF7, MDA-MB-231 and normal airway cells at 50 µg/ml after 48 h incubation. Data are presented as a mean of 3 biological repeats ± SE, and statistically analyzed relative to DMSO control with one-way ANOVA and followed by a Tukey HSD means comparison test (*P < 0.05, ***P < 0.001, NS – non-significant). (D) Representative microscopy image of A549, NCI-H460 and normal airway cell lines treated with DMSO or 50 µg/ml VLC80 and stained with Hoechst (blue) and PI (violet). Scale bars represent 100 µm. (E) A549 cells were incubated for 24 h with VLC80 at 20, 50, and 100 µg/ml and apoptosis was assessed relative to DMSO control by staining with Annexin-V/PI and analyzing the apoptotic rate via flow cytometry. The lower left quadrant contains the double negative population of vital cells, the lower right quadrant contains the early apoptotic stage (Annexin V + /PI −) population, the upper right quadrant contains the dead (annexin V + /PI +) cells population, and the upper left quadrant contains non-apoptotic dead cells (annexin V − /PI +).