Fig. 7: p27 regulates autophagosome positioning via the control of ATAT1 stability.

A HEK 293 cells were transfected with p27 and/or ATAT1-GFP for 24 h. p27 was immunoprecipitated and p27-bound ATAT1 was detected with anti-GFP antibodies. Expression levels of p27 and ATAT1-GFP were determined by immunoblotting in extracts. β-actin was used as loading control. B HEK 293 cells were transfected with 1 µg of ATAT1-GFP vector and increasing amounts of p27 for 24 h. Expression levels of ATAT1-GFP and p27 were determined by immunoblotting. β-actin was used as loading control. C Representative images of cells from (B) acquired with the IncuCyte. Green fluorescence represents ATAT1 expression. Scale bars are 200 µm. D Quantification of experiments as described in (C). Fluorescent object confluence was measured and normalized to cell confluence (phase contrast). Values were normalized to empty vector transfected cells condition. n = 9 images per condition from three independent experiments. E HEK 293 cells were transfected with 1 µg ATAT1-GFP for 48 h and 2 µg p27 or empty vector and treated with 50 µg/ml cycloheximide (CHX) for 16 h. Images were acquired with an IncuCyte every 4 h to monitor ATAT1-GFP expression levels (green fluorescence). Values of fluorescent object confluence were normalized to cell confluence. n = 12 images per time point for each condition from three independent experiments were used for quantification. F Representative images of experiment as described in (E). Scale bars are 200 µm. G Validation of ATAT1 siRNA. HEK 293 cells were transfected with Myc-ATAT1 for 24 h and then with 50 nM siRNA for another 48 h and subjected to immunoblot against Myc. Actin was used as loading control. H p27^+/+ and p27^−/− MEFs were transfected with either control or ATAT1 siRNA. After 24 h, cells were collected and fixed (0 h) or glucose starved for 24 h. Cells were stained for LC3B, acetylated α-tubulin and α-tubulin. Scale bars are 50 µM. I Acetylated (Ac-) α-tubulin and α-tubulin immunoblot on p27^+/+ MEFs transfected with ATAT1 siRNA and collected after 48 h. Since tubulin levels change after ATAT1 siRNA transfection, β-actin was used as loading control. J Mean percentage of cells with over 50% of perinuclear LC3B⁺ vesicles from three (p27^+/+) or two (p27^−/−) experiments as described in (H). Autophagosome distribution was evaluated based on a percentage of LC3B integrated density in perinuclear area (designated as region of interest [ROI]) versus LC3B integrated density in the whole cell. Number of images used for quantification: p27^+/+: n = 31 (siControl), n = 34 (siATAT1); p27^−/−: n = 22 (siControl), n = 22 (siATAT1). At least 105 cells per condition were analyzed per experiment. D, E, J Graphs show means ± SEM. Statistical significance was determined by 1-way (D) or two-way ANOVA (E, J) followed by Bonferroni multiple comparison test.