Figure 1.
Induction of cell death by violacein in intrinsically resistant TF1 myeloerythroid leukaemia cells.
(A) TF1 cells are unusually resistant with respect to PCD. Cells were exposed for 24 hrs to FasL, TNFα and mitoxantrone. (B) TF1 cells were exposed to various concentrations of violacein (X-axis) in the presence of 10% FCS and analysed for cellular survival (as assayed by the capacity of cellular cultures to reduce MTT; Y-axis) 24 hrs, 48 hrs and 72 hrs later as indicated. (C) Analysis of cellular DNA content by FACS shows that violacein-induced TF cell death is accompanied by breakdown of the genome. Each value represents the mean ± SEM of three independent experiments.
Figure 2.
Induction of cell death by violacein does not progress through canonical modes of programmed cell death.
(A) Cell death induced in TF1 cells by a 24 hrs treatment with 2 µM of violacein is not sensitive to inhibitors of pro-apoptotic caspases. (B) The autophagosome inhibitor 3-methyladenine does not impair violacein-induced cellular suicide in TF1 cells. (C) Apparent absence of a necrotic effect of violacein in TF1 cells, as assayed by the ethidium bromide/acridine orange assay. (D) Western blot analysis of beclin-1 and autophagy-specific LC3B isoform levels excludes a major role of autophagy in violacein effects in TF1 cells. Nevertheless, violacein clearly increases levels of cleaved PARP and thus cell death induction by violacein represents a mode of PCD.
Figure 3.
Induction of cell death by violacein is associated with an ultrastructurally unique programme of cellular demise.
(A) Using transmission electron microscopy, the ultrastructural effects of violacein-induced cell death were characterised at different time points following application of the indole derivative. No apparent morphological evidence for induction of either autophagy, apoptosis, necroptosis or pyroptosis is obtained, as for instance the integrity of the mitochondria and plasma membrane is maintained until the final stages of cellular suicide and no increase in the number of autophagosomes is seen. Highly distinctive of violacein-induced cell death, however, is the endoplasmic reticulum and Golgi linearization and at later time points nuclear mottening. (B) High magnification examples of endoplasmic reticulum and Golgi linearization, characteristic for violacein effects 20 hrs following application of the chemopreventive indole.
Figure 4.
Induction of cell death by violacein is associated with a major rearrangement of cellular biochemistry.
(A) The PKB-mediated survival cassette is not inhibited by 2 µM violacein treatment in TF1 cells. Although Western Blot analysis of signalling intermediate phosphorylation does not perfectly correlate with the kinase enzymatic activity, it is obvious that also phosphorylation status analysis does not provide evidence for diminished survival signalling following TF1 cell stimulation. Note that GSK3β activity is negatively regulated by its PKB-mediated phosphorylation (B) Viability of TF1 cells was evaluated in the presence of Calpeptin (50 µM) or after violacein (2 µM) treatment. (C) Plotting correlation between phosphorylation of specific peptide substrates shows that 30 minutes following violacein treatment only minor changes in the cellular kinome are observed. More long-term treatment, however, causes major remodelling of the kinome. The value in the graph gives the Pearson product. (D) Heatmap of significantly altered kinases in TF1 cells in response to violacein treatment. (E) Venn diagram depicting the distribution of phosphorylated spots at different time points (30 min is depicted in blue, 16 hrs in yellow and 24 hrs in green). (F) Comparison between statistically significant phosphorylated spots in TF1 cells treated with 2 µM violacein for 0 hrs with cells treated for 16 hrs and 24 hrs. This graph shows the correlation between fold change and p-values for the statistically significant phosphorylated spots at the different time points of treatment with violacein.