Fig. 2.

The LIR motif within OPTN and NDP52 is essential for efficient mitophagy. a, b Penta KO HeLa cells with or without untagged Parkin expression rescued by stable expression of the indicated GFP-tagged receptors were analysed by immunoblotting after 21 h incubation with OA (a) for quantification of the remaining CoxII levels (b). c–h, penta KO HeLa cells stably expressing mtKeima, untagged Parkin and either GFP-OPTN (c), GFP-OPTN(F178A) (d), GFP-NDP52 (f) or GFP-NDP52(V136S) (g), were imaged by live-cell confocal microscopy ((c, d, f, g); GFP-receptor channels provided in Supplementary Fig. 1g, h), and analysed by Fluorescence Activated Cell Sorting (FACS) to quantify the percentage of 561 nm mtKeima-positive cells (e, h), after time-course incubation with OA. (Representative FACS plots provided in Supplementary Fig. 1j; FACS analysis and representative images for penta KO & GFP-p62 provided in Supplementary Fig. 1e, f, i). Data in (b) are mean ± s.d. from four independent experiments. Data in (e, h) are mean ± s.d. from three independent experiments. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001 (b one-way ANOVA; e, h two-way ANOVA). ns: not significant. Scale bars: 10 µm