Fig. 2: P56S-VAPB alters late step of autophagy.

a–c Immunoblot analysis of transiently transfected control GFP/RFP, wt-VAPB and P56S-VAPB; in NSC-34 cells (a), NIH3T3-GFP-LC3 fibroblasts (b) and of the Primary fibroblasts isolated from autophagy reporter GFP-LC3 transgenic mice (MEF-GFP-LC3) (c), and then additionally treated with the autophagy inhibitor Bafilomycin A (2 µM) for 2 h to inhibit autophagy followed by treatment of Rapamycin (2 µM) for 4 h to accelerate autophagy (a) or Bafilomycin A (2 µM) alone (b, c). Note the unchanged LC3-II levels in P56S-VAPB transfected cells after Rapamycin or Bafilomycin A treatment. Corresponding densitometric data are shown at the bottom; where the numbers represent the relative band intensity of LC3-II/LC3-I, normalised with tubulin levels. Graph pad prism, unpaired Student’s t test for comparison between two sample groups. Values were expressed as mean ± standard error of mean (SEM) from three independent experiments. The asterisks denote significant differences (*p < 0.05, ***p < 0.001), while # denotes absence of a significant difference. d, e NIH3T3 fibroblasts stably expressing RFP-GFP-LC3 was transfected either with HA tagged wt-VAPB or P56S-VAPB. In all, 24 h later the fusion of autophagosomes with lysosomes was measured by live cell imaging for additional 12 h (d), the rate of autophagosome fusion reflected by the Pearson coefficient (green/red fluorescence ratio) at each time point indicated (e). Values are represented as means ± S.E.M. of triplicate experiments *p ≪ 0.0001. f Co-labelling of wt-VAPB and P56S-VAPB transfected HEK-293 as well as HeLa cells with STX17, showing its marked reduction (arrows) in cells harbouring P56S-VAPB aggregates. Scale bar: 10 µm. g Immunoblot analysis of STX17 proteins in HEK-293 as well as HeLa cells, transiently transfected with control GFP, wt-VAPB and P56S-VAPB. Note the significantly reduced levels of STX17 in P56S-VAPB overexpressing cells. Unpaired Student’s t test for comparison between two sample groups. Values were expressed as mean ± standard error of mean (SEM) from three independent experiments. The asterisks denote significant differences (*p < 0.05). h EM analysis of P56S-VAPB transfected NSC-34 cells showing massive accumulation of autophagosomes (white arrows, quantification) in the vicinity of several lysosomes (black arrows). For the quantification of autophagosomes, numbers of large, membrane-bound autophagosomes, filled with the cargoes were counted manually from 20 to 30 cells from both the wt-VAPB as well as P56S-VAPB. The asterisks denote significant differences (***p < 0.001). Scale bars: 1 µm.