Fig. 6. Vps34 lactylation promotes endosome-lysosomal degradation.

(A) The Rab5 and EEA1 localization in U2OS cells (control shRNA and Vps34 KD/rescued by vector, Vps34^WT, and Vps34^2KR). Rab5 and EEA1 colocalized on enlarged endosomes in Vps34 delactylation cells. Scale bar, 10 μm. (B) EGFR degradation assay. HEK293T cells (control shRNA and Vps34 KD/rescued by vector, Vps34^WT, and Vps34^2KR) were treated with EGF (100 ng/ml) for 0,1.5, and 3 hours. EGFR were analyzed by Western blot. EGFR levels were quantified by ImageJ software and normalized by percentage of 0-hour EGFR level. (C) Cathepsin D maturation assay in U2OS cells (control shRNA and Vps34 KD/rescued by vector, Vps34^WT, and Vps34^2KR) was analyzed by Western blot with Cathepsin D to show procathepsin D, preprocathepsin, and mature cathepsin D. (D) The levels of procathepsin D and preprocathepsin D were quantified by ImageJ software. Data are shown as means ± SD; **P < 0.01, n = 3. (E) Percentage of Rab7 and LysoTracker colocalization. Data are shown as means ± SD; **P < 0.01, n = 30. (F) Rab7 and LysoTracker colocalization in U2OS cells (control shRNA and Vps34 KD/rescued by vector, Vps34^WT, and Vps34^2KR) was analyzed by confocal microscopy. Scale bar, 10 μm. (G) Lysosome staining assay by confocal microscopy with Lamp1 antibody in U2OS cells (control shRNA and Vps34 KD/rescued by vector, Vps34^WT, and Vps34^2KR). Scale bar, 10 μm. (H). Lamp1 expression in U2OS cells (control shRNA and Vps34 KD/rescued by vector, Vps34^WT, and Vps34^2KR) was analyzed by Western blot.