Fig. 4

CEP downregulates MYO1C and inhibits the interaction/colocalization of MYO1C with LC3 and LAMP1. MDA-MB-231 cells were treated without or with CEP (4 μM) for 24 h, after which whole cell lysates were digested using trypsin and subjected to LC-MS to identify differentially expressed proteins. Each group consist three biological repeats. a 36 cytoskeleton related proteins upregulated or downregulated at least 2-fold were selected and then divided into seven group by DAVID Functional Annotation Bioinformatics Microarray Analysis. b Cells were treated with various concentrations of CEP for 24 h, or treated with CEP (4 μM) for different time intervals as indicated. The expression of MYO1C was determined by western blot analysis. c Cells were treated with CEP (4 μM) or Rapa (0.25 μM) or Baf (20 nM) for 24 h, after which the expression of MYO1C was detected by western blot analysis. d MDA-MB-231 cells were treated without or with CEP (4 μM) or Rapa (0.25 μM) for 24 h. Whole-cell lysate was prepared and subjected to immunoprecipitation using anti-LC3 and the associated LC3B-II and MYO1C were determined using immunoblotting. e The colocalization of LC3 and MYO1C was determined by confocal microscopy. f The Pearson’s correlation coefficient (R²) of MYO1C and LC3 colocalization 50 cells of three independent experiments (mean ± SD, *P < 0.05; **P < 0.01). g LAMP1 was immunoprecipitated, and the expression MYO1C was determined by western blot analysis. h The fluorescent images showed the colocalization of MYO1C with LAMP1. i The Pearson’s correlation coefficient (R²) of MYO1C and LAMP1 colocalization was from 50 cells of three independent experiments (mean ± SD, *P < 0.05; **P < 0.01)