|
Fig. 2 VPS41 variants contribute to impaired lysosomal functions in vitro.(A) Immunocytochemistry assessing TFE3 in rescue experiments using transient expression of wild-type (WT) and mutant VPS41-t2a-GFP in VPS41 KO ESC. Transfected cells are marked by GFP and GFP+ cells showing no TFE3 relocalization to the cytoplasm are marked with white asterisks. Nuclei are counterstained with DAPI. Scale bars = 50 µm. (B) Quantification of A counting a minimum of 80 GFP+ cells from three replicates. ***P = 0.0001, binomial test (expected distribution based on relocalization upon transfection with the wild-type construct). (C) Western blotting detecting VPS41 (endogenous VPS41 99 kDa, V5-tag-VPS41 105 kDa, input 30 µg) and LAMP2 (100–120 kDa, input 20 µg), in wild-type and VPS41 KO ESCs and VPS41 KO ESCs transiently rescued with wild-type or mutant VPS41. (D) Quantification of C, *P < 0.05; **P < 0.01; ***P < 0.001 one-way ANOVA, multiple comparison test of mutant constructs to the wild-type. (E) Percentage of GFP+ cells upon transient transfections of VPS41-KO cells with VPS41-GFP plasmid spiked with mCherry, expressing wild-type or mutant VPS41. ***P = 0.0006, ****P < 0.0001, one-way ANOVA, multiple comparison test of mutant constructs to the wild-type; >17 000 cells were analysed per sample.