Fig. 4

STO-609 treatment reduces p-AMPK, increases p62, and blocks autophagic flux in spheroids. a Cell lines (EOC cells: iOvCa147-MA, OVCAR8 and COV318; non-malignant cells: FT190) were seeded into 6-well ULA plates to form spheroids for 24 h prior to treatment with 10 μM STO-609, or DMSO as a vehicle control. Protein lysates were isolated at 24 h post-treatment. Immunoblot analysis was performed for p-AMPK (T172), AMPK, p62, and LC3B; tubulin served as a loading control. b Densitometric analysis for p-AMPK/AMPK, p62/tubulin and LC3-II:I ratio from the immunoblots for the three EOC cell lines together were tested by Student’s t-test. Asterisks indicate significant differences relative to control (*, p < 0.05; ****, p < 0.001). c Representative phase contrast and fluorescence images of DMSO- and STO-609-treated spheroids using OVCAR8-mCherry-eGFP-LC3B, COV318-mCherry-eGFP-LC3B, and FT190-mCherry-eGFP-LC3B cells. Treatments were performed as described above and images were captured after 24 h. Scale bar = 200 μm