Fig. 3: Parkin translocation is induced by Calcineurin in the absence of PINK1. | Cell Death & Differentiation

Fig. 3: Parkin translocation is induced by Calcineurin in the absence of PINK1.

From: Activation of Ca2+ phosphatase Calcineurin regulates Parkin translocation to mitochondria and mitophagy in flies

Fig. 3

A Representative confocal images of PINK1 KO MEF cells transfected with mCherry-Parkin, mito-YFP and with empty vector (EV) or constitutively active CaN (ΔCnA). B Quantification of A Graph bar shows mean ± SEM of percentage of cells with mCherry-Parkin on mitochondria for at least ≥ 300 cells per biological replicate. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 3-4; p < 0.001). C Quantification of A using Squassh. The graph bars show mean ± SEM of Squassh colocalization coefficient for at least ≥ 50 images per biological replicate. 0 = no colocalization, 1 = perfect colocalization. At least 3 independent experiments were performed. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 3-4; p < 0.001). D Representative confocal images of PINK1 KO MEF cells transfected with mCherry-ParkinS65E, UbS65E, mito-YFP and with empty vector (EV) or dominant negative CaN (ΔCnAH151Q). E Quantification of D Graph bar shows mean ± SEM of percentage of cells with mCherry-Parkin on mitochondria for at least ≥ 300 cells per biological replicate. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 4; p < 0.0001). F Quantification of D using Squassh. The graph bars show mean ± SEM of Squassh colocalization coefficient for at least ≥ 50 images per biological replicate. 0 = no colocalization, 1 = perfect colocalization. Two-way ANOVA followed by Tukey’s multiple comparison test (n = 4; p < 0.0001). G Parkin thermal stability assay. WT MEFs expressing constitutive active CaN (ΔCnA) or empty vector (EV) were suspended in PBS and snap-freezed in liquid nitrogen before being aliquoted into a PCR strip and incubated at the indicated temperature for 3 min. The lysates were centrifugated at high speed and the soluble fraction was loaded into SDS-PAGE gel. Representative Western blotting analysis for Parkin stability is shown. H Densitometric analysis of G. Student’s t-test (n = 4; p < 0.05). I Parkin thermal stability assay. PINK1 KO MEFs expressing constitutive active CaN (ΔCnA) or empty vector (EV) were suspended in PBS and snap-freezed in liquid nitrogen before being aliquoted into a PCR strip and incubated at the indicated temperature for 3 min. The lysates were centrifugated at high speed and the soluble fraction was loaded into SDS-PAGE gel. Representative Western blotting analysis for Parkin stability is shown. J Densitometric analysis of I. Student’s t-test (n = 6; p < 0.05).

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