Research Paper Advance Articles

Figure 3. Reduced mitochondrial NAD⁺ in WRN depleted cells. (A, B) WRN-KD in the HEK293-mitoPARP reporter cell line led to decreased mitochondrial NAD⁺ compared to Scr as measured by PAR signal relative to β-Actin. Cells were grown in the presence of the PARP inhibitor 3-AB (1 mM) and collected at the designated timepoints after removal of 3-AB from the media (0, 3, 6 h). (A) Quantification of 6 biological repeats. Scramble (Scr) is paired with WRN-KD from the same biological repeat with a black line in the graph. Statistical significance was found between 0 h (right after the release of 3-AB treatment) compared to 3 h and 6 h (Two-way ANOVA, Tukey’s multiple comparisons test. Scr (Veh): 0 h to 3 h and 6 h p-values < 0.0001. WRN-KD (Veh): 0 h to 3 h p-value = 0.001; 0 h to 6 h p-value < 0.0001). Further increased PARylation signal, resembling increased mitochondrial NAD⁺ levels, was observed in Scr between 3 h and 6 h after 3-AB release (Two-way ANOVA, Tukey’s multiple comparisons, p-value = 0.0030), however no change was observed in WRN-KD cells between 3 h and 6 h, suggesting less available mitochondrial NAD⁺ (Two-way ANOVA, Tukey’s multiple comparisons, p-value = 0.8310). Moreover, there was a significant reduced level of PAR in WRN-KD cells compared to Scr at 6 h after 3-AB release (Two-ANOVA, Tukey’s multiple comparisons, p-value = 0.0321). (B) Representative western blot of PAR and β-Actin signal. (C, D) WRN-KD leads to decreased colony formation in HEK293 cells (Two-way ANOVA, Tukey’s multiple comparisons, p-value = 0.012), which was not rescued by overexpression of the human mitochondrial NAD⁺ transporter SLC25A51 or 1 mM NR treatment for 24 h. Overexpression of SLC25A51 in HEK293 (HEK293-SLC25A51) did on the other hand significantly increase colony formation in Scr cells (Two-way ANOVA, Tukey’s multiple comparisons, p-value = 0.0049). Colony formation assay was performed for Scr or WRN-KD (30 nM siRNA) in three different HEK293 cell lines: Parental HEK293, HEK293-mitoPARP and HEK293-SLC25A51. The HEK293-mitoPARP cells were exposed to 6 h 3-AB release before seeding out for colonies in medium containing 1 mM 3-AB for 11 days. (C) Representative images of stained colonies. (D) quantification of 3 biological replicates with 3 technical repeats. (E) Confirmation of WRN-KD was shown to be 90–100% in all biological replicates. Western blotting of GFP-PARP1 and FLAG confirmed the specific expression of the mitoPARP and SLC25A51-constructs, respectively. The dotted line indicates that the representative blots are from two different blots with their respective loading controls. Statistics were performed with GraphPad Prism using Two-way ANOVA with Tukey’s multiple comparison.