Fig. 3

CEP inhibits autophagic degradation in MDA-MB-231 and MCF-7 cells by interfering autophagosome-lysosome fusion. a MDA-MB-231 and MCF-7 cells were exposed to various concentrations of CEP for 24 h. The expressions of autophagy-related proteins (p-ULK1(Ser757), Beclin-1, ATG5, and ATG7) were detected by western blot. b Cells were treated without or with CEP (4 μM) in the presence or absence of 20 nM bafilomycin A₁ (Baf) or 0.25 μM rapamycin (Rapa) for 24 h, the expressions of SQSTM1 and LC3B-I/LC3B-II were analyzed by western blot. The ratio of LC3-II/LC3-I was quantified in three independent experiments (mean ± SD, *P < 0.05 or **P < 0.01). c Cells were transfected with a tandem reporter construct (tfLC3), and were exposed to CEP (4 μM), Baf (20 nM) and Rapa (0.25 μM). The colocalization of EGFP and mRFP-LC3 puncta was examined by confocal microscopy. Scale bars: 10 μm. d Cells were treated with CEP (4 μM) or Baf (20 nM) for 24 h. The fluorescent signals were detected by confocal microscopy after stained with LysoTracker Red. Scale bars: 10 μm. e Cells were treated with CEP (4 μM) or Rapa (0.25 μM) for 24 h, whole-cell lysate was prepared and subjected to immunoprecipitation using anti-LC3B and the associated LC3B-II and LAMP1 were determined using immunoblotting. f Cells cotransfected with mRFP-LC3 and LAMP1-mGFP were treated without or with CEP (4 μM), Baf (20 nM), or Rapa (0.25 μM). The colocalization of mRFP-LC3 and LAMP1-mGFP was detected by confocal fluorescent microscopy. Scale bars: 10 μm. g The Pearson’s correlation coefficient (R²) of LAMP1 and LC3 colocalization was from 50 cells of three independent experiments (mean ± SD from, n.s, not significant **P < 0.01). h Cells were exposed to various concentrations of CEP for 24 h, or treated with CEP (4 μM) for different time intervals as indicated. The expressions of LAMP1, LAMP2 were determined by western blot and GAPDH was used as a loading control