Fig. 1: Autophagy regulation by ACBP in Saccharomyces cerevisiae.

a, b Immunoblotting analysis of protein extracts from wild type (WT) and ∆acb1 cells expressing a GFP-Atg8 fusion protein. Blots were probed with antibodies against GFP to detect GFP-Atg8 and free GFP, which is indicative of autophagic flux, and against GAPDH as loading control. Representative results (a) and densitometric quantification (b) at 1 and 2 days are shown. (n = 4). c Relative alkaline phosphatase (ALP) activity at 1, 4, and 6 days of chronological aging of WT and ∆acb1 cells expressing Pho8pΔN60 (n = 3). d, e Fluorescence microscopy of WT and ∆acb1 cells expressing a GFP-Atg8 chimera at day 2 of chronological aging. Propidium iodide (PI) counterstaining served to visualize dead cells. Scale bar = 5 μm. Autophagic cells were defined as cells with clear vacuolar GFP fluorescence and depicted as percentage of viable (PI⁻) cells. Per strain and replicate, 500–650 cells were manually counted. (n = 5). f, g ALP activity of WT and ∆acb1 cells expressing Pho8pΔN60 at the indicated times of chronological aging with or without 40 nM rapamycin (Rapa) (f) or upon nitrogen starvation (−N) for 4 h and 24 h (g) (n = 3–5). Quantitative results are reported as means ± SEM. Symbols indicate statistical (Student’s t-test) comparisons with controls (n.s, not significant; *p < 0.05, ***p < 0.001).