Figure 3 | Scientific Reports

Figure 3

From: Parallel damage in mitochondrial and lysosomal compartments promotes efficient cell death with autophagy: The case of the pentacyclic triterpenoids

Figure 3The alternative text for this image may have been generated using AI.

Consequences of autophagic flux inhibition.

(a) LC3 lipidation (LC3-II form) after 6 hours of treatment with DMSO (control) and triterpenoids (20 μM) (i). Bars indicated the mean of relative amount of LC3-II (%) normalized by the baseline expression of GAPDH (ii). (b) Keratinocytes treated with DMSO (control) and triterpenoids (20 μM) were immunostained with LC3-II at indicated times (6 or 24 hours). Fold change of LC3-II compared to control were represented as arbitrary units. (c) At T1 following staining of lysosomes (LTR), cells treated with DMSO (control), triterpenoids (20 μM) or TEM (15 μM) were immunostained for LC3-II (blue) and for lysosomal marker LAMP2A (green). At bottom, plot profile (LC3-II/lysosome/LAMP2A) of lines drawn in the images. (d) At T1 HaCaT treated with DMSO (control) and triterpenoids (20 μM) in presence or absence of BAF (2 ηM). Following staining of lysosomes with LTR (red), cells were immunostained for LC3-II (blue) and for COXIV (green). Fluorescence plot profiles represented line scans of LTR-loaded lysosomes and LC3-II/COXIV. (e) At T1 lysosomal enzymes were extracted from subcellular fraction (lysosomes), following fluorescence assays for detection of cathepsins L (CTSL) and B (CTSB) activities. (f) CTSB activity from lysosomal fraction as a function of time. (g) At T2 HaCaT treated with DMSO (control) and triterpenoids (20 μM) following lysosomal staining with LTR (red) and immunostaining for total CTSB (green). Plot profile (CTSB/lysosome) of lines drawn in the images (i) and surface plots (ii). At T2 with DMSO (control) and BA (20 μM) total CTSB (ρg/mL) was assayed by ELISA (iii). T1 = after treatment for 24 hours; T2 = 24 hours after T1; T3 = 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. Significance difference (p < 0.05) was depicted by asterisk.

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