Fig 1: h-Prune KD HEK293 cells show decreased levels of polyP. (A) Significant immunoblots and densitometric analysis that demonstrate decreased levels of h-Prune in HEK293 cells after transfection with siRNA. Note that transfection with scrambled siRNA does not induce any significant changes in the levels of h-Prune. In the densiometric analysis, circles represent individual values from samples treated with lipofectamine; and squares and triangles individual values of samples transfected with Scrambled siRNA and h-Prune siRNA, respectively. (B) Graph showing the levels of polyP in our cellular models. This quantification was conducted by measuring the fluorescence of DAPI-polyP. The levels of polyP are decreased by knocking down Prune. Transfection with the scrambled siRNA did not induce any significant effects on the levels of polyP. (C) The plausible kinase activity of h-Prune was also assayed. However, no variations in the levels of polyP were found in this case either. PPK was used as a positive control, and h-Prune was incubated either in the PPK or the PDE buffer. The results are shown as mean ± SEM. The PPK assay (as it is an enzymatic study) was conducted using experimental triplicates, while the other experiments were conducted using at least experimental and biological triplicates. * p ≤ 0.05, ** p ≤ 0.01.
Fig 2: h-Prune KD deleteriously affects mitochondrial respiration, decreasing the cellular levels of ATP. Using Oroboros 2k respirometry, ATP-linked respiration (left) and maximal respiration (center) were assayed in HEK293 cells. Our data show that these two parameters are decreased in h-Prune KD, compared to the control samples. Moreover, the cellular levels of ATP (right), which were analyzed using luminescence, are also decreased when h-Prune is knocked down. The results are shown as mean ± SEM of at least three independent experiments. * p ≤ 0.05, *** p ≤ 0.001.
Fig 3: Recombinant h-Prune does not hydrolyze short (13–33 Pi)- and medium (45–160)-chain polyP, even though it conserves its ability to release 5′AMP from cAMP. (A) Graph showing that recombinant h-Prune conserves its PDE activity. To conduct this in vitro study, we assayed the release of 5′AMP from cAMP. Increasing concentrations of h-Prune with 10 mM MgCl2 were tested. PDE was used as a positive control, and assay buffer as a negative control. (B) h-Prune is not able to hydrolyze short (13–33 Pi) and medium (45–160 Pi) chains of polyP after 24 h of the assay in the presence of 5 mM MgCl2. These chain lengths are those that should be more common in mammalian cells. The levels of polyP were assayed using DAPI fluorescence, and ALP was used as positive control. The results are expressed as mean ± SEM. The PDE assay (as it is an enzymatic study) was conducted using experimental triplicates, while the other experiments were conducted using at least experimental and biological triplicates. *** p ≤ 0.001.
Fig 4: In HEK293 cells, the effects of h-Prune on the levels of polyP are mediated by the regulation of the activity of ATP synthase, but not by an effect on the expression of ATP synthase, or on the regulation of mitochondrial membrane potential or mitochondrial biomass. (A) Significant images obtained after loading our cells with TMRM. Note that no major differences are found between control and h-Prune KD HEK293 cells. FCCP 10 μM was used as a positive control to induce mitochondrial depolarization. (B) Assay of the activity of ATP synthase in our samples. Note that decreased levels of the activity of this enzyme are found in h-Prune KD, compared to the levels shown in the control samples. (C) Significant immunoblots and densitometric analysis that corroborate that h-Prune KD HEK293 cells have significantly decreased levels of the protein. Moreover, using the same methods, our data show that the levels of ATP5 are increased in h-Prune KD, while those of TOM20 are not affected by knocking down Prune. In the densiometric analysis, circles represent individual values from samples treated with lipofectamine, while triangles represent individual values from h-Prune KD samples. The results are shown as mean ± SEM of at least three independent experiments. Scale bar: 50 μM. * p ≤ 0.05, ** p ≤ 0.01.
Fig 5: The effects of h-Prune KD on the levels of polyP are conserved between humans and Drosophila. (A) Phylogenetic analysis of the protein sequences shows that the Prune orthologs are highly conserved between humans and Drosophila. This is especially significant in the PPX consensus sequence, which is marked in red in the sequence alignment. The numbers on the right are the residues on the remaining C-terminal fragment. (B) Assay of the levels of polyP (using DAPI-polyP fluorescence) in homozygous (pn1/pn1) and heterozygous mutants. Results are shown as mean ± SEM of at least three independent experiments. * p ≤ 0.05, ** p ≤ 0.01.
Supplier Page from Abcam for Recombinant Human PRUNE protein (GST tag N-Terminus)