Figure 2
Analysis of mitochondrial membrane impairment in HaCaT.
Keratinocytes were treated with DMSO (control), triterpenoids (20 μM), or CCCP (2 μM). (a) After 3 hours, the decrease in mitochondrial inner transmembrane potential (ΔΨm) was measured in terms of Rh123 fluorescence intensity relative to control (100%) (i). The decrease in Rh123 fluorescence intensity was evaluated as a function of time (ii). (b) Following staining for lysosomes with LTG (green) and mitochondria with MTR (red), cells were treated for 6 hours. Line scans (at bottom) indicated association between lysosome and mitochondria. This relationship represented the lines drawn in the images. (c) Keratinocytes were treated with DMSO (control), triterpenoids (20 μM). Following staining of lysosome with LTR (red), cells were immunostained for LC3-II (blue) and for the mitochondrial marker COXIV (green) at T1 (i). Bars showed the average of fluorescence intensity from multiple images (ii). (d) Scatter-plots of HaCaT following immunostaining for LC3-II (FL1) and COXIV (FL3) at T1 (i). Mitochondria mas (COXIV) compared to control and represented as arbitrary units (ii). (e) Scatter-plots at T1 showing LC3-II and COXIV in treated cells in presence or absence of BAF (20 ηM) added at the last two hours of the treatment. Keratinocytes treated with DMSO (control), triterpenoids (20 μM), or CCCP (2 μM) for 6 hours, following (f) citrate synthase (CS) activity represented as arbitrary units after ponderation to control; and (g) immunostaining for COXIV (green) and Parkin (red). At bottom, plot profile (Parkin/COXIV) of lines drawn in the images. T1 = after treatment for 24 hours; T2 = at 24 hours after T1; T3 = at 48 hours after T1. 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.
