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Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: Lipid accumulation in human breast cancer cells injured by iron depletors

Fig. 3

Vacuoles remain distinct from autophagosomes. a Immunofluorescence localisation of LC3 in vacuolated MDA-MB-231 cells. Cells were treated with DFO or Dp44mT for 48 h, immunostained with anti-LC3 (red) and with anti-tubulin (green) antibodies, and then analysed by confocal microscopy. Blue, DAPI. The cell population shifts in size and granularity and becomes heterogeneous after DFO or Dp44mT treatments. This is an excerpt of the results obtained from several (at least three) experiments. There was little overlap between the phase-lucent vacuoles and compartments labelled with markers for lysosomes (LysoTracker) or autophagosomes (LC3II), suggesting a defect at the late endosome-lysosome boundary. b Western blotting analysis of LC3-I and LC3-II expression in treated cells for 48 h and the relative Ponceau-stained filter used as a loading control. c Lysotracker staining analysis by flow cytometry analysis. Results are expressed as mean of the far red (APC-A) fluorescence intensity or forward-scatter (FSC-A) factors (x-axis) and of the side-scatter (SSC-A) factor (y-axis), i.e. granularity and size, respectively, of untreated and DFO- or Dp44mT-treated MDA-MB-231 cells for 48 h, showing the increased SSC-A in DFO-treated cells. DMSO-treated cells have lower signal intensity. d Confocal microscopy analysis of induced vacuoles sequestering lucifer yellow (LY). Differential interference contrast (DIC) images and LY fluorescence (green) of control, DFO-, and Dp44mT-treated cells. Not all vacuoles were sufficiently acidic to massively incorporate LY. Dp44mT-treated cells show a diffuse staining pattern throughout the cytoplasm, which could correspond to some degree of intracellular membrane permeabilisation. This is an excerpt of the results obtained from several (at least three) experiments. Scale bars: 100 μm

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