Figure 1 | Cell Death & Disease

Figure 1

From: Activation of RARα induces autophagy in SKBR3 breast cancer cells and depletion of key autophagy genes enhances ATRA toxicity

Figure 1

ATRA induces autophagy in SKBR3 cells. (a) SKBR3 and MDA-MB-453 breast cancer cells were treated either with 0.1 or 1 μM ATRA for 2 and 4 days, respectively. β-Catenin and LC3B levels were measured by western blotting. GAPDH was used as a loading control. (b) LC3B-II quantification from at least three independent experiments. LC3B-II expression was normalized to GAPDH and to vehicle-treated SKBR3 cells. (c) β-Catenin staining of SKBR3 and MDA-MB453 cells. Cells were control or ATRA (1 μM) treated for 2 days. β-Catenin FITC (fluorescein isothiocyanate) and nuclear DAPI (4',6-diamidino-2-phenylindole) staining as analyzed by confocal microscopy are shown. (d) Quantification of endogenous LC3B puncta. LC3B in SKBR3 and MDA-MB453 cells treated with 1 μM ATRA for 2 days. (e) LC3B-II western blotting and quantification of the autophagic activation upon treatment with 1 μM ATRA for 2 days in the presence or absence of BafA for 2 h at 200 nM. LC3B-II levels were normalized to GAPDH and to vehicle-treated SKBR3 cells. Standard deviations for five independent experiments are shown. (f) Long-lived protein degradation assay of SKBR3 and MDA-MB453 cells treated as in (e). Radioactivity was determined by liquid scintillation counting of at least three independent experiments. Absolute proteolysis is shown. (g) Long-lived protein degradation assay of SKBR3 treated as in (e) and including treatment with the macroautophagy-specific inhibitor 3-MA (10 mM). Analysis as in (f). Mann–Whitney U-test: *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001

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