Figure 3

RARα but not RARβ and RARγ agonists induce autophagic flux in SKBR3 cells. (a) SKBR3 cells were treated with 1μM of RARα, RARβ or RARγ agonists for 2 days in the presence and absence of BafA during the last 2 h, before subjection to immunofluorescence for LC3B. (b) Quantification of LC3B puncta from the experiment described in (a). Three independent experiments were quantified as described in Schläfli et al.⁴¹ (c) Autophagic flux was determined from the immunofluorescence analysis shown in (a). N=3, Student's t-test and **P<0.01. (d) Long-lived protein degradation assay of control or cells treated with 1 μM of RARα, RARβ, RARγ agonists in the presence or absence of 200 nM BafA. Radioactivity was determined by liquid scintillation counting of four independent experiments. Data are shown as BafA-sensitive proteolysis. (e) Western blot analysis for VE-cadherin, β-catenin and LC3B of SKBR3 cells treated as in (a). (f) LC3B-II levels on western blot were normalized to GAPDH and quantified from at least five independent experiments using the ImageJ software (NIH, Bethesda, MD, USA). LC3B-II levels of control treated cells were arbitrarily set to 100%. Mann–Whitney U-test: *P<0.05, **P<0.01 and ****P<0.0001