Figure 5. Suppression of LC3 and BECN1 lowers cellular capacity to overcome proteotoxicity.

(a) The five genes most lost in the autophagy KEGG pathway in OV compared with 20 other cancers in tumour gene loss prevalence. (b) Log₂ SNP6 array scores for each tumour in OV compared with three OV cell lines. ‘Genome' corresponds to the average gene score for an individual tumour. OVCAR3 is the most established OV cell line with high-grade serous genetics³⁰, whereas IGROV1 and SKOV3 are ovarian cancer cell lines without serous OV genetics. (c) OVCAR3 has delayed accumulation of acidic vacuoles including autophagosomes and lysosomes, as measured by acridine orange flow cytometry, when treated with the autophagy/lysosome inhibitor chloroquine (10 μM). Data represent the mean±s.e.m. from four independent experiments. Note that additional cell lines are tested in Supplementary Fig. 15. (d) Western blots of autophagosomal Lc3-II indicate reduced accumulation of autophagosomes in OVCAR3 and increased levels of ER stress marker Grp78 when treated with chloroquine. Lysates from three independent experiments were analysed and a representative blot is shown. (e) SKOV3 cells knocked down by BECN1 and LC3 shRNA were treated with 10 μM chloroquine for the indicated times. Only shLC3 showed reduced accumulation of autophagosomes by flow cytometric reading of acridine orange stain. Data represent the mean and s.e.m. from four independent experiments. (f) Western blots of cells treated as in e, showing reduced Lc3-II accumulation only in shLC3 cells. Lysates from three independent experiments were analysed and a representative blot is shown. (g) OV cells were treated with chloroquine for 48 h at the doses indicated and stained for cell loss by crystal violet. Data represent the mean±s.e.m. from eight independent experiments. *P<0.05, ***P<0.001 by two-tailed Student's t-test.