Fig. 2

PRKAA1/2 knockdown does not alter LC3-II and p62 levels in spheroids yet blocks autophagic flux. a Double knockdown of both AMPK α1 and α2 catalytic subunits was performed by co-transfection of PRKAA1 and PRKAA2 siRNA in adherent iOvCa147-MA and OVCAR8 cells; non-targeting siRNA (siNT) served as a control. At 72 h post-transfection, cells were trypsinized and seeded into 6-well ULA plates for 48 h. Immunoblot analysis was performed for p-AMPK (T172), AMPK, p62, and LC3B; tubulin served as a loading control. b Densitometric analysis for AMPK/tubulin, p62/tubulin, and LC3-II:I ratio from the immunoblots were tested for significance using a Student’s t-test (****, p < 0.001). c OVCAR8 mCherry-eGFP-LC3B cells were transfected with siNT and siPRKAA1/2 as described above and seeded into 24-well ULA plates. Phase contrast and fluorescence images were captured at 48 h post-seeding. Scale bar = 200 μm. d Quantification of eGFP (green markers) and mCherry (red markers) fluorescence intensity per spheroid (normalized to spheroid area) in siNT and siPRKAA1/2-transfected OVCAR8-mCherry-eGFP-LC3B cells was performed using Image J software and tested for significance by two-way ANOVA followed by Sidak’s multiple comparison test (**, p < 0.01; ****, p < 0.001)