VDAC1 and MICU1 analysis. (A) RT-PCR analysis demonstrating expression of vdac1 at 1, 24, 48 and 72 hpf. (B) RT-PCR analysis of vdac1 in wt controls and after MO mediated vdac1 k/d, demonstrating a marked effect of the vdac1 MO on vdac1 wt transcript levels. (C) Dopaminergic (DA) neuronal cell count in wt, wt vdac1 (wt injected with MO against vdac1), pink1−/−, pink1−/− vdac1 (pink1−/− injected with MO against vdac1) at 3 dpf, reflecting the lack of an effect of VDAC1 inactivation on DA neuronal cell count in pink1−/− (P > 0.99) (*P = 0.0105). (D) qPCR analysis of micu1, showing significant upregulation of micu1 in pink1−/− larvae at 3 dpf compared to wt (**P = 0.0066). (E) qPCR analysis of micu2, showing a non-significant down regulation of micu2 in pink1−/− larvae at 3 dpf compared to wt (nsP = 0.0902). (F) qPCR analysis of mcur, showing a non-significant upregulation of mcur in pink1−/− larvae at 3 dpf compared to wt (nsP = 0.2088). (G) qPCR analysis of emre, showing a non-significant down regulation of emre in pink1−/− larvae at 3 dpf compared to wt (nsP = 0.2712). (H) RT-PCR analysis of micu1 at 3 dpf in uninjected larvae (WT) and after MO mediated k/d (micu MO), demonstrating the marked effect of micu1 MO injection on micu1 mRNA transcript levels. (I) Dopaminergic (DA) neuronal cell count in wt, wt micu1 (wt injected with MO against micu1), pink1−/−, and pink1−/− micu1 (pink1−/− injected with MO against micu1) zebrafish larvae at 3 dpf, demonstrating the rescue effect of MICU1 inactivation on DA neurons in PINK1 deficiency (***P = 0.0004).
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