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. Author manuscript; available in PMC: 2013 Apr 1.
Published in final edited form as: Nat Cell Biol. 2012 Sep 30;14(10):1024–1035. doi: 10.1038/ncb2589

Figure 1. Loss of myosin VI function leads to an accumulation of autophagosomes.

Figure 1

(a) Western blot analysis of myosin VI depleted Hela cells untreated or treated with 100 nM Bafilomycin A1. (b) Quantitation of Western blot LC3-II intensity (+/− s.d.) (n=3). **p<0.01, ***p<0.001 (c) Confocal immunofluorescence microscopy of LC3 punctae (green) in Hela cells following knockdown of myosin VI. Hoechst labels nuclei (blue). Scale bar, 20 μm. (d) Quantitation of LC3- and p62-positive punctae was performed and results are represented as average punctae fluorescence intensity/cell (+/− s.d.) (n=3) from >1500 cells/experiment. (e) Western blot analysis of parental Hela cells or Hela cells stably expressing siRNA resistant GFP-myosin VI transiently transfected with a single myosin VI siRNA oligonucleotide following treatment with 1 μM MG132. (f) Western blot analysis of mouse embryonic fibroblasts cultured from wild-type and Snell’s Waltzer (SV) mice treated with 1 μM MG132 or 100 nM Bafilomycin A1. (g) Quantitation of Western blot LC3-II intensity (+/− s.d) (n=3). **p<0.01. (h) To evaluate effects on autophagosome biogenesis, results are displayed as the fold increase in normalised LC3-II intensity with Bafilomycin A1 compared to untreated control. (+/− s.d.) (n=3) (i) Western blot analysis of cortical neurons from wild-type and SV mice treated with 1 μM MG132 in the absence or presence of 100 nM BafilomycinA1. (j) Quantitation of Western blot LC3-II intensity was performed. (n=2) (k) To evaluate effects on autophagosome biogenesis, results are displayed as fold increase in normalised LC3-II intensity with Bafilomycin A1 compared to untreated control. (n=2) (l) Confocal immunofluorescence microscopy of mock or myosin VI siRNA treated Hela cells stably expressing RFP-GFP-LC3 reporter. Hoechst labels nuclei (blue). (m) Quantitative data of RFP and GFP signal overlap from confocal images of mock or myosin VI siRNA treated Hela cells expressing RFP-GFP-LC3. Data is represented as the Pearson’s coefficient of RFP and GFP signal correlation from >100 cells/experiment. (+/− s.d.) (n=3) Scale bar, 20 μm. (n) Confocal immunofluorescence microscopy of myosin VI depleted RPE cells stably expressing GFP-LC3 immunostained against GFP (green) and Cathepsin D (red). Hoechst labels nuclei (blue). Insets represent magnified boxed regions. Scale bar, 20 μm.