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. 2014 Jan 20;33(4):282–295. doi: 10.1002/embj.201385902

Figure 4.

Figure 4

Parkin-dependent MDVs are targeted to lysosomes independently of autophagy
  1. HeLa cells, transfected with siRNA targeting Drp1 and GFP-parkin (green), were treated with 25 μM antimycin A for 90 min following a 30-min pretreatment with 50 nM bafilomycin A1, then fixed and immunostained against PDH E2/E3 bp (red) and TOM20 (blue). PDH E2/E3 bp-positive/TOM20-negative MDVs colocalizing with GFP-parkin (arrows) or not (circles) are indicated. Scale bar, 30 μm.
  2. Quantification of PDH E2/E3bp-positive/TOM20-negative vesicles in HeLa cells treated with antimycin A in the presence of the lysosomal inhibitors bafilomycin A1 or pepstatin A and E-64d; both total number (white bars) and the number colocalizing with GFP-parkin (gray bars) are indicated. Bars represent the mean ± s.e.m. P-values are given for total vesicle number (n = 19 to 120 cells in 2–3 experiments; **P < 0.01; ***P < 0.001).
  3. HeLa cells, transfected with GFP-parkin (green) and siRNA targeting Drp1 and Atg5, were treated with 25 μM antimycin A for 90 min, then fixed, immunostained for PDH E2/E3 bp (red) and TOM20 (blue), and counterstained for Hoescht (gray). PDH E2/E3 bp-positive/TOM20-negative MDVs colocalizing with GFP-parkin (arrows) or not (circles) are indicated. Scale bars, 20 μm.
  4. Quantification of PDH E2/E3 bp-positive/TOM20-negative vesicles from HeLa cells transfected with the indicated siRNA; both the total number (white bars) and the number colocalizing with GFP-parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P-values are given for the total vesicle number (n = 42–64 cells in 2–3 experiments); ns, not significant.