Fig. 3.

OPTN and NDP52 recruitment is influenced by LIR-mediated interactions with Atg8s. a Representative immunofluorescence images of penta KO HeLa cells stably expressing untagged Parkin rescued by expression of the indicated GFP-tagged receptors; immunostained for Atg13, HSP60 and GFP after treatment with OA for 3 h. (Equivalent images for TAX1BP1 & NBR1 are provided in Supplementary Fig. 5b). b–g Automated image analysis of the average Atg13 foci count per cell (b, e), Atg13 foci volume (c, f) and intensity of foci in the GFP channel (d, g), in penta KO HeLa cells stably expressing untagged Parkin, rescued by stable expression of either GFP-OPTN/GFP-OPTN(F178A) (b–d) or GFP-NDP52/GFP-NDP52(V136S) (e–g), immunostained for Atg13, HSP60 and GFP after time-course incubation with OA. (Data from other time points shown in Supplementary Fig. 2g-j). h, i, j, l Representative immunofluorescence images (h, i) and automated image analysis of GFP-tagged receptor translocation (j, l) in penta KO HeLa cells stably expressing untagged Parkin after rescue expression of either GFP-OPTN/GFP-OPTN(F178A) (h, j) or GFP-NDP52/GFP-NDP52(V136S) (i, l), immunostained for HSP60 and GFP after time-course incubation with OA (times indicated). k, m Automated image analysis of GFP-OPTN (k) or GFP-NDP52 (m) translocation in WT and hexa KO HeLa cells stably expressing untagged Parkin, after time-course incubation with OA and immunostaining for HSP60 & GFP. (Representative images for (k, m) provided in Supplementary Fig. 2q, r). Data in (b–g) and (j–m) are mean ± s.d. from three independent experiments. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001 (two-way ANOVA). ns: not significant. Scale bars: overviews, 10 µm; insets, 2 µm