Fig. 9: Calcineurin requires PINK1 to induce mitophagy in MEFs.

A Representative Western blot analysis of protein lysates extracted from PINK1 WT and PINK1 KO MEFs. Cells of the indicated genotype were transfected with constitutive active CaN (ΔCnA) or empty vector (EV). Graph bars shows mean ± SEM of indicated mitochondrial protein level normalized to Actin. Student’s t-test (n = 8-11; *p < 0.05; **p < 0.01). B Representative confocal images of cells of the indicated genotype transfected with constitutive active CaN (ΔCnA) or empty vector (EV). Cells were pretreated with proteasome inhibitor MG132 before being fixed, permeabilized and incubated with the indicated primary antibodies and corresponding fluorophore-conjugated secondary antibodies. C Quantification of B using Squassh. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 4; p < 0.05). D Representative confocal images of MEFs transfected with constitutive active CaN (ΔCnA) or empty vector (EV), and with the GFP-TAB2 NZF sensor for K63-linked ubiquitin chain, and mitoRFP to visualize the mitochondrial network. In one group 10μM CCCP was used as a positive control to trigger Parkin translocation and Parkin-dependent ubiquitination of mitochondria. E Quantification of D. Graph bars shows the co-localisation of k63 linked ubiquitin probe (GFP-TAB2 NZF) with mitochondria. Co-localisation index was analysed with Coloc2 plugin (Fiji). tM2 = fraction of GFP TAB2 NZF co-localising with mitochondria (0 = null, 1 = 100%). F Representative Western blot analysis of protein lysates extracted from PINK1 WT and PINK1 KO MEFs, and incubated with the indicated antibodies, after being immunoprecipiated with HisPur Ni-NTA Magnetic Beads (Thermoscientific). Cells of the indicated genotype were transfected with His-Ubiquitin and constitutive active CaN (ΔCnA) or empty vector (EV). Cells were pretreated with proteasome inhibitor MG132 for 4 h before being collected.