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. 2015 Jul 27;5:12425. doi: 10.1038/srep12425

Figure 1. Analysis of biological effects of BA and OA.

Figure 1

(a) Survival rates at T3 as a function of dose (10–30 μM). (b) At T1 with DMSO (control) and triterpenoids (20 μM), HaCaT keratinocytes were stained with the dyes Acridine Orange (AO) and Propidium Iodide (PI), and evaluated under microscope. Arrows indicated nuclear PI positive dead cells with acidic vacuoles accumulation. (c) Alternatively, at T2 with DMSO (control), triterpenoids (20 μM) or TEM (15 μM) the acidic vacuoles AO-stained were quantified by FACS. (d) At T2 HaCaT keratinocytes treated with DMSO (control) and triterpenoids (20 μM) following staining with Neutral Red (30 μg/mL) were evaluated under microscope (i). At indicative times, the cell survival was assayed by MTT and linearly correlated to lysosomal content measured in terms of autophagy arbitrary units – AAU (ii). (e) At T3 with DMSO (control) and triterpenoids (20 μM) the cell survival of HaCaT and HeLa treated cells was assayed by MTT reduction following AAU calculation. (f) At T1 and T2, HaCaT keratinocytes treated with DMSO (control) and triterpenoids (20 μM) following FACS were gated according the Side Scatter (SSC) and Forward Scatter (FSC) parameters (i). After gating, bars showed the ponderation of cell size (FSC) and granularity (SSC) compared to control and represented as arbitrary units (ii). (g) After the same experimental condition (f), a pseudo-color scatter-plots showed gating of HaCaT treated cells according to two parameters (mitochondrial mass and cell death), following staining with MitoTracker green FM and PI dye inclusion (i). Regarding these parameters, subpopulations of treated-cells were identified, weigthed by control and represented as arbitrary units (ii). All results were obtained from at least three independent experiments and expressed as mean values ± standard error. Multiple statistical comparisons were calculated by ANOVA test, and the p-value for each pairwise group was determined by Dunnett’s T3 (high variance between groups) or Bonferroni (low variance between groups) post-hoc test. The analysis of correlation was done using Pearson’s coefficient (r). Significance difference (p < 0.05) was depicted by asterisk.