Fig 2: Summary.HIF-1 transcriptionally activates HEY1. HEY1 in turn transcriptionally represses PINK1. PINK1 is important to mitochondrial biogenesis. During hypoxia, ROS accumulates at the mitochondria due to inefficient electron transfer through the electron transport chain in the mitochondria, HIF-1 overcomes the oxidative stress through elevating HEY1 to reduce PINK1-mediated mitochondrial biogenesis, making HCC cells less dependent on mitochondria to reduce mitochondrial ROS production. Therefore, the HIF-1/HEY1 pathway confers survival advantages to HCC cells which frequently experience hypoxia.

Fig 3: The half-life of the p.I368N mutant PINK1 protein is reduced. a-b HeLa cells were simultaneously transfected with PINK1 siRNA and DNA constructs encoding for PINK1-V5 WT or p.I368N mutant and incubated with 100 μg/ml cycloheximide (CHX) alone (a) or pretreated with 10 μM CCCP for 2 h before incubation with cycloheximide (b). Cell lysates were analyzed by WB and V5/vinculin ratios were normalized to PINK1-V5 WT. Shown is the mean ± SEM from three independent experiments. Half-life (t1/2) of PINK1-V5 proteins was determined by curve fitting (one phase exponential decay). c Co-immunoprecipitation (IP) of the HSP90/CDC37 chaperone complex and the import channel of the mitochondrial outer membrane TOM40 with PINK1-V5. HeLa cells were transfected with PINK1-V5 WT, p.I368N or another PD-mutant PINK1 p.L347P as a control and left untreated or treated with 10 μM CCCP for 2 h. PINK1-V5 protein complexes were immunoprecipitated from cell lysates using V5 antibody conjugated to agarose. Representative WB is shown for input and IP samples probed with antibodies against V5 (PINK1), HSP90, CDC37, and TOM40. GAPDH served as a loading control. d Quantification of the co-immunoprecipitated proteins as performed in (c) is shown as the protein/V5 ratio. Data represent mean ± SEM from six independent experiments. Statistical significance was assessed by two-way ANOVA with Tukey’s post hoc; ***, p < 0.0005; **, p < 0.005; *, p < 0.05

Fig 4: p.G411S, but not p.Q456X heterozygotes show persistently impaired PINK1 kinase activity under endogenous conditions over time. Primary human skin fibroblasts were left untreated or stressed with 1 µM of the potassium ionophore valinomycin (VM) for the indicated times to depolarize mitochondrial membranes. Two cell lines per genotype were analysed. PINK1 genotypes of individuals are stated as wild-type (WT), heterozygous (HET) or homozygous (HOMO) along with the respective PINK1 mutation. (A) Representative western blots with total cell lysates were analysed for levels of PINK1, phosphorylated ubiquitin (p-Ser65-Ub), parkin, and its substrate mitofusin 2 (MFN2). Closed and open triangles indicate unmodified MFN2 and its ubiquitinated forms, respectively. Anti-vinculin was used as loading control. (B and C) Quantification of protein levels from western blots of six independent experiments. Shown is the mean per genotype ± SEM. Statistical significance was assessed with two-way ANOVA, Tukey’s post hoc test (*P < 0.05; **P < 0.005; ***P < 0.0005). (B) Shown are levels of PINK1 and p-Ser65-Ub normalized to wild-type at 24 h. Levels of total, unmodified ubiquitin were comparable from all cells and at all times (data not shown). p.Q456X heterozygotes showed significantly reduced levels of PINK1, concomitant with initially decreased levels of p-Ser65-Ub. However, p.G411S showed PINK1 levels similar to wild-type, but significantly reduced levels of p-Ser65-Ub over time. (C) Shown are relative levels of parkin and MFN2 normalized to 0 h valinomycin treatment. Absolute levels of parkin were similar in wild-type and all heterozygous PINK1 mutant cells, but were significantly elevated in p.Q456X homozygotes; however, no difference was seen for MFN2 (data not shown). (D) Quantitative MSD assay in 96-well plate format of p-Ser65-Ub levels from cell lysates. Cells were treated with 1 µM valinomycin (VM) for the indicated times, lysed and p-S65-Ub was captured with phospho-specific ubiquitin antibodies and detected with total ubiquitin antibodies. Shown is the mean per genotype ± SEM normalized to wild-type at 24 h from three independent experiments. Statistical significance was assessed with two-way ANOVA, Tukey’s post-hoc test (*P < 0.05; *** P < 0.0005). (E) Quantitative high content imaging in 96-well plate format of p-Ser65-Ub intensity in primary human fibroblasts before and after valinomycin (VM) treatment. Shown is the mean per genotype ± SEM normalized to wild-type at 0 h and 24 h from three independent experiments. Statistical significance was assessed with two-way ANOVA, Tukey’s post hoc test (*P < 0.05; **P < 0.005; ***P < 0.0005).

Fig 5: Effect of PDE2A2 inhibition on adipocyte browning.a Representative western blot and b quantification of PINK1 in primary white adipocytes treated with forskolin or BAY-60 in the presence of the mitochondrial uncoupler CCCP with or without stimulation of the beta-adrenergic receptor with Isoprenaline (ISO). The amount of PINK1 is normalized to Calnexin and is shown as fold change relative to levels in CCCP-treated cells. Data are mean ± s.e.m, N = 5. c mRNA expression of nuclear-encoded mitochondrial and thermogenic genes in primary white adipocytes treated with 1 μM NE, 1 μM BAY-60 or 1 μM NE + 1 μM BAY-60. mRNA level is shown as the fold change relative to levels in non-treated cells, N = 7. d Representative western blot and e quantification of UCP1 from primary white adipocytes treated with 1 μM NE, 1 μM BAY-60 or 1 μM NE + 1 μM BAY-60. The amount of UCP1 is shown as the fold change relative to levels in non-treated cells. N = 4. f mRNA expression of mitochondria-encoded genes in primary white adipocytes treated with 1 μM NE, 1 μM BAY-60 or 1 μM NE + 1 μM BAY-60. mRNA level is shown as the fold change relative to levels of Hprt N = 5. g UCP1-dependent oxygen consumption of primary white adipocytes treated with with 1 μM NE, 1 μM BAY-60 or 1 μM NE + 1 μM BAY-60, N = 6. h Representative images (left) and quantification (right) of the average lipid droplet size of primary white adipocytes treated with with 1 µM NE, 1 µM BAY-60 or 1 µM NE + 1 µM BAY-60, N = 5. All values are expressed as mean ± s.e.m. One-way ANOVA and Tukey’s multiple comparison tests were performed for (c, e–h). Student’s t-test statistical analysis was performed for (b). *P < 0.05; **P < 0.01; ***P < 0.005; ns = not significant.