Figure 4 | Cell Death & Disease

Figure 4

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

Figure 4

Depletion of RARα prevents ATRA-induced autophagic flux in SKBR3 cells. (a) β-Catenin, VE-cadherin and GAPDH western blot analysis of SKBR3 cells transduced with a scrambled shRNA (CTRL) or RARα-specific shRNA (shRARα) treated with ATRA for 2 days in the presence and absence of 200 nM BafA during the last 2 h. (b) Immunohistochemistry of β-catenin in control and shRARα-transduced cells subjected to 1 μM ATRA for 4 days. Pictures were taken on a confocal microscope. (c) LC3B qPCR analysis of control and ATRA-treated SKBR3 cells expressing a scramble shRNA or shRARα. Raw Ct values were normalized to ABL-1 mRNA and to the vehicle-treated control cells (ΔΔCt method). (d) LC3B western blotting and quantification of the autophagic activity in control and RARα-knockdown SKBR3 cells upon treatment with 1 μM ATRA for 2 days in the presence or absence of BafA for 2 h at 200 nM. LC3B-II expression was normalized to GAPDH and to vehicle-treated control cells. Standard deviations from at least five independent experiments are shown. (e) Long-lived protein degradation assay of control and ATRA-treated SKBR3 cells transduced with a scrambled shRNA (CTRL) or shRARα in the presence or absence of BafA (200 nM) or 3-MA (10 mM) during the chase phase. Radioactivity was determined by liquid scintillation counting in three independent experiments. Mann–Whitney U-test: *P<0.05, **P<0.01 and ***P<0.001

Back to article page