Fig. 1: p27 promotes autophagy flux in glucose-deprived cells. | Cell Death & Disease

Fig. 1: p27 promotes autophagy flux in glucose-deprived cells.

From: p27 controls autophagic vesicle trafficking in glucose-deprived cells via the regulation of ATAT1-mediated microtubule acetylation

Fig. 1

A LC3B immunoblot in p27+/+ and p27−/− MEFs in full medium (0 h) or glucose-deprived for 48 h ± CQ (50 µM) for 2 h before collecting cells. Actin was used as loading control. B LC3 turnover, measured as a ratio of LC3B-II signal intensity in starved cells in the presence of CQ versus LC3B-II under the same conditions without CQ, as described in A. Graph shows means ± SEM from n = 6 independent experiments. C Autophagosome formation was evaluated by measuring the ratio of LC3B-II levels, in the presence of CQ to block lysosomal degradation, at 48 h and 0 h of glucose starvation. Graph shows means ± SEM from n = 3 independent experiments. D p27+/+ and p27−/− MEFs were glucose-starved for the indicated times and stained for p-ATG16L1 and LC3B. DNA was stained with Hoechst 33342. E Graph shows the average number of p-ATG16L1 puncta per cell ± SEM. Number of cells used for quantification: p27+/+: n = 210 (0 h), 625 (24 h), n = 822 (48 h); p27−/−: n = 222 (0 h), n = 906 (24 h), n = 284 cells (48 h). F Graphs show mean ± SEM of the distribution of autophagophores (p-ATG16L1+/LC3B−, red), early autophagosomes (p-ATG16L1+/LC3B+, yellow) and mature/sealed autophagosomes (p-ATG16L1−/LC3B+, green) in cells deprived of glucose for 24 h (p27+/+: n = 15; p27−/−: n = 27 images and 48 h (p27+/+: n = 16; p27−/−: n = 19 images). G Immunoblot for LC3B and p27 in mCherry-eGFP-LC3B MEFs used in experiment described in (H). In the LC3B immunoblot, the 75 kDa band is mCherry-GFP-LC3B and the ~13 kDa doublet is endogenous LC3B. β-tubulin was used as loading control. H p27+/+ and p27−/− MEFs expressing mCherry-eGFP-LC3B were glucose deprived for 48 h and fixed prior to microscopy analysis. CQ treatment for 2 h was used as negative control. Scale bars are 50 µm. I Quantification of experiments as described in (H) in MEFs glucose starved for 48 h. Yellow LC3B dots represent autophagosomes, while red LC3B dots represent autolysosomes. Graph shows mean ± SEM number of autolysosomes and autophagosomes from n = 3 independent experiments. At least 150 LC3B dots were analyzed per conditions per experiment. B, C, E, F, I Statistical significance was evaluated by two-tailed Student’s t test with Welch’s correction (B, C) or 2-way ANOVA (E, F, I).

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