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Figure 5


Fig. 5. (a) Quantification of GFP-expressing COS-7 cells with aggregates and abnormal nuclear morphology transiently transfected with dominant-negative dynamin II (DN-Dyn-II) or empty vector control and EGFP-tagged huntingtin exon 1 containing 74 polyglutamine repeats (3:1 ratio) for 48 hours. ***P<0.0001. (b) Distribution of GFP-LC3 vesicles in control (left) or DN-dynamin (red) transfected cells (right). (c) COS-7 cells were transiently transfected with empty vector (Control), DN-dynamin (DN-Dyn) and GFP-LC3 (3:1 ratio) for 24 hours. GFP-positive cells with an increased number of LC3-positive vesicles (>20 vesicles per cell) were quantified; ***P<0.0001. (d) Lysates from HeLa cells expressing empty vector control (Cont) or DN-dynamin (DN-Dyn) were blotted for endogenous LC3, and actin as control. Quantification of band intensities from four independent experiments is shown; **P<0.001. Under exposure conditions that allow endogenous LC3-II quantification in these cells, the LC3-I signal is frequently too low to be detected. (e) NRK cells were transiently transfected for 15 hours with either empty vector [control; with or without bafilomycin A1 (BafA1)] or DN-dynamin (DN-Dyn), and mRFP-LC3 and GFP-lgp120. mRFP-LC3 and GFP-lgp120 double-stained vesicles in individual cells are given in percent (Jahreiss et al., 2008). ***P<0.0001. (f) HeLa cells transfected with control vector or DN-dynamin (DN-Dyn) together with HA-Atg12 and Atg5 were subjected to western blot analysis using anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. Atg5-Atg12:Atg12 ratio from three independent experiments is shown. (g) Quantification of GFP-expressing COS-7 cells with aggregates and abnormal nuclear morphology transiently transfected with dominant-negative (DN) Vps4 or empty vector control and EGFP-tagged huntingtin exon 1 containing 74 polyglutamine repeats (3:1 ratio) for 48 hours. ***P<0.0001.





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