Figure 2.
Follicular lymphoma-associated VMA21 mutations activate autophagic flux. (A) HEK293T cells transfected with empty vector, WT, or mutant (93X) VMA21 treated -/+ bafilomycin A1. Immunoblots of the indicated antigens. (B) Densitometry quantification (LC3-II:ACTB) of n = 4 independent experiments from representative panel A. Statistical comparisons: i) VMA21 93X versus WT (A; lanes 5–6). ii) VMA21 93X versus WT plus -/+ bafilomycin A1. (A; lanes 8–9), using unpaired 2-tailed t-testing and Bonferroni corrections (*: p < 0.05; **: p < 0.01). Bars: standard deviations. (C) Electron microscopy and enumeration of autophagosomes (AP), autolysosomes (AL) and late endosomes/lysosomes (LE/LY) in HEK293T cells expressing VMA21 WT or MUT 93X. Representative images of N = 70–90 each. (D) Statistical comparisons of data generated in panel C using unpaired 2-tailed Mann-Whitney U-test and Bonferroni corrections. ***: p < 0.001). Bars: Standard eviations. (E-K) S. cerevisiae cells expressing either Vma21 (WT) or Vma21[∆66-77] were analyzed for autophagy. The cells were grown in YPD (-N, 0 min) to mid-log phase, and shifted to nitrogen starvation (SD-N) (E) The GFP-Atg8 processing assay. Pgk1 was used as a loading control. (F) The ratio of free GFP to Pgk1 after 45 min SD-N treatment was quantified. Mean ± SD of n = 4 independent experiments. Unpaired, 2-tailed t-test; *: p < 0.05. (G) Pho8∆60 enzymatic activity was measured under growing and nitrogen-starvation conditions. Mean ± SD of n = 5 independent experiments. Two-way ANOVA; ***: p < 0.001. (H) The prApe1 processing assay. Dpm1 was used as a loading control. (I) The ratio of Ape1 to prApe1 after 15 min SD-N treatment was quantified. Mean ± SD of n = 3 independent experiments. Unpaired, 2-tailed t-test; *: p < 0.05. (J) The Atg8 lipidation assay. Pgk1 was used as a loading control. (K) The ratio of Atg8–PE to Pgk1 after 45 min of nitrogen starvation was quantified. Mean ± SD of n = 4 independent experiments. Two-way ANOVA; *: p < 0.05, **: p < 0.01.