Fig. 1: Yeast Set2 and human SETD2 act as positive regulators of autophagy.

a, b Wild-type (WT; WLY176) and set2Δ (YAB418) yeast strains were grown in rich-medium conditions until mid-log phase. Cells were shifted to a nitrogen-starvation medium (-N) with and without PMSF that blocks vacuolar protein degradation for the indicated timepoints. a Protein extracts were analyzed by immunoblotting and incubated with anti-Atg8 and anti-Pgk1 (loading control) antisera. Quantification of Atg8–PE:total Atg8 ratio is shown in panel b. c, d Human cervical carcinoma HeLa cells were transfected with a pool of siRNAs targeting SETD2, or as control scramble siRNAs, for 48 h. Treatment with bafilomycin A₁ (BafA1, 40 nM) for 4 h, which inhibits the fusion of autophagosomes with lysosomes, was used to block the autophagy flux. c Cells were harvested, and protein extracts analyzed by immunoblotting for LC3 and ACTB/β-actin (used as a loading control). The quantification of LC3-II (i.e., LC3–PE) levels normalized to ACTB is depicted in panel d and shows a significant decrease of the LC3-II:ATCB ratio in SETD2-deficient cells treated or not with BafA1. e Post-transfection with the indicated siRNA pool, cells were treated with 250 nM torin1 for 2 h or left untreated. MAP1LC3B expression was monitored by immunofluorescence analysis and LC3 puncta per cell quantified. f–h HeLa cells were transfected with an expression vector encoding SETD2, or mock-transfected with the corresponding pcDNA3.1 empty vector used as a control, for 24 h. f Cells were harvested, and protein extracts analyzed by immunoblotting for LC3 and ACTB (used as a loading control). The quantification of LC3-II (i.e., LC3–PE) levels normalized to ACTB is depicted in panel g and shows a significant increase of the LC3-II:ATCB ratio in SETD2-overexpressing HeLa cells. h Post-transfection with the indicated plasmids, cells were treated with 250 nM torin1 for 2 h or left untreated. MAP1LC3B expression was monitored by immunofluorescence analysis and LC3 puncta per cell quantified. b Bars display the mean of three independent experiments, error bars represent SD. d, g Bars display the mean of at least three independent experiments (d, n = 5; e, g, and h n = 3), error bars represent SEM; *P < 0.05; **P < 0.01; ***P < 0.001.