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Fig. 2 | Journal of Ovarian Research

Fig. 2

From: Beclin-1 expression is retained in high-grade serous ovarian cancer yet is not essential for autophagy induction in vitro

Fig. 2

Autophagy is readily inducible in ascites-derived cells. a Spheroids filtered from patient ascites fluid for immunofluorescence analysis (n = 3 EOC samples). Nuclei (blue) and cytoplasmic LC3 staining (green) are visible. Orange arrowheads indicate LC3 puncta. Scale Bar: 20 μm. b EOC cells were seeded to non-adherent plates and lysates obtained at indicated time points. Immunoblot of EOC67 (top) and quantification of LC3-I and LC3-II expression relative to Actin (n = 3 EOC samples) were performed. Bars: Mean ± SEM; *p < 0.05, **p < 0.01. c. Quantifications also performed on immunoblots of parallel adherent (ADH) and (SPH) cultures of multiple EOC samples (n = 10) at 24 h (top) and 72 h (bottom). Bars: Mean ± SEM; *p < 0.05. d Parallel adherent (~80 % confluent) and spheroid cultures were analyzed by transmission electron microscopy. Orange arrowheads denote autolysosomes (autophagosomes that have fused with lysosomes). Scale bars: 500 nm. e Adherent (ADH) cultures (~80 % confluent) of the indicated samples were subjected to 24 h of Akti-1/2 treatment (EOCs: 5 μM; iOvCa147-E2: 3.5 μM) and protein lysates generated. Parallel spheroid cultures (24 h under non-adherent conditions) of the indicated samples were also simultaneously lysed and immunoblot performed for indicated proteins

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