Supplementary Figure 9: Atypical ubiquitin chain linkages signal for mitophagy.

(A) Immunostaining of HeLa cells co-transfected with GFP-Parkin and mitochondrial-localized, FLAG-tagged DUBs. 24 h after transfection cells were immunostained for FLAG (green) and endogenous HSP60 (red). Each DUB co-localizes with HSP60 indicating proper mitochondrial targeting (see merge). In addition, expression levels appear uniform across all DUBs transfected. Representative cells are shown for each transfection. Scale bar, 10 μm. (B) Large field images from Fig. 5a. Solid yellow outlines highlight cells with GFP-Parkin and DUB-Flag expressing cells. Dashed yellow outlines highlight cells expressing only GFP-Parkin. Scale bar, 30 μm. (C) Immunostaining of HeLa cells co-transfected with GFP-Parkin and HA-tagged ubiquitin variants as listed in Fig. 5c. Only GFP-Parkin expressing cells are outlined. Solid lines indicate TOM20 positive staining cells; dashed lines indicate TOM20 negative staining cells. Scale bar, 20 μm. (D) Quantification of TOM20 in HeLa cells treated with CCCP (15 μM, 24 h) and transfected with either beta-Gal or HA-ubiquitin. A significant difference in TOM20 was observed with a 24 h CCCP treatment preventing analysis of individual HA-ubiquitin mutants tested in Fig. 5c. Each point represents a single experiment with a mean intensity from 25–40 cells measured; the line represents the mean from n = 5 experiments and error bars represent s.e.m. Significance is reported as one-Way ANOVA with Holm–Sidak’s multiple comparison test (^∗P < 0.05).