Figure 2. The autophagic machinery is activated and it interacts with overexpressed AURKA.

(A) Left: HEK293 cells imaged by transmission electron microscopy. Lower panels correspond to the magnified dotted area. Cells were transfected with control- or AURKA-specific siRNAs, or with an AURKA–GFP vector (AURKA O/E). m = mitochondria; magenta arrows = lysosomes. (Right) Quantification of lysosomal number per 10 μm² of cell surface. n = 20 images per condition from two independent experiments. Scale bar: 1 μm. (B) Relative LysoTracker Red fluorescence measured by flow cytometry on HEK293 cells transfected as indicated. n = 3 independent experiments with at least 30,000 cells per condition quantified. Data represent means ± SEM, A.U., arbitrary units. (C, D) Förster’s Resonance Energy Transfer by Fluorescence Lifetime Imaging Microscopy analyses on MCF7 cells expressing AURKA–GFP (C) or AURKA ΔNter–GFP (D) alone or together with mCherry–MAP1LC3B or LAMP1–mCherry. Dotted area: autophagosome/autolysosome-rich areas. Pseudocolour scale: pixel-by-pixel ΔLifetime. Lower panels: corresponding ΔLifetime quantifications in the dotted area. n = 10 cells per condition of one representative experiment (of three). (E) Graphical representation of AURKA–GFP or AURKA ΔNter-GFP ΔLifetime values in the presence of mCherry MAP1LC3, plotted as a function of the red/green intensity ratio. n = 30 cells per condition from three independent experiments. Scale bar: 10 μm. Data extend from minimum to maximum, unless where indicated. (A, B, C, D) ***P < 0.001 against the “Control siRNA” (A), the “Empty vector” (B), or the “AURKA-No acceptor” (C, D) conditions. NS, not significant.