Fig. 4. ESK981 induces accumulation of lysosomes through inhibition of autophagic flux in prostate cancer cells.

(a) Representative TEM micrographs of DU145 cells after 300 nM ESK981 treatment for 24 hours from three independent experiments. Micrograph of ESK981-treated cell shows mostly clear vacuoles adjacent to an autophagic vacuole, which is magnified in the red dashed box. Red arrow indicates a mostly clear vacuole. N, nucleus.

(b) Representative micrographs of MDA-PCa-146-12 PDX tumors taken by TEM after five days of treatment from three independent experiments. Red arrows indicate vacuoles in ESK981 group, and yellow arrows indicate cellular materials inside the vacuole. N, nucleus.

(c) Representative immunofluorescence staining of LAMP1 in DU145 cells treated with control or 300 nM ESK981 for 24 hours from three independent experiments.

(d) Lysosomal activity was quantified by FACS after staining with LysoTracker Green. VCaP, LNCaP, PC3, and DU145 cells were treated with increasing concentrations of ESK981 for 24 hours (left). VCaP, LNCaP, PC3, and DU145 cells were treated with DMSO, ESK981 (300 nM), bafilomycin A₁ (100 nM), or ESK981-bafilomycin A₁ combination for 24 hours (right).

(e) Ratio of GFP/RFP signal in PC3 and DU145 GFP-LC3-RFP-LC3ΔG stable expressing cells with the indicated treatment for 24 hours. Data were analyzed by two-tailed unpaired t test from four independent experiments and presented as mean ± SEM. *p<0.05; **p<0.01.

(f) Paired MEF cells with either Atg5 wild type (Atg5^+/+) or Atg5 knockout (Atg5^−/−) were treated with 300 nM ESK981 for 24 hours. Representative morphologies are shown in phase contrast microscopy (left) from three independent experiments. ATG5 and LC3 protein levels were examined by western blot (right).