Research Paper Advance Articles

Figure 4. WRN is indispensable in proliferation as mitochondrial or cellular NAD⁺ augmentation is unable to reinstall compromised proliferation in immortalized WRN-KD cells. (A, B) WRN-KD led to decreased colony formation in HEK293 parental cells (Two-way ANOVA, Tukey’s multiple comparisons, p-value = 0.0009). The proliferation defects caused by knockdown were not rescued by either 1 mM NR treatment throughout the colony formation period or overexpression of SLC25A51. Media was replaced with fresh media every 3 days. (A) Representative images, acquired with Bio-Rad Chemidoc. (B) Quantification of 3 biological replicates with 3 technical repeats/replicates. Statistical analysis was done using two-way ANOVA with Tukey’s multiple comparisons test. (C) Western blotting confirming WRN-KD of 90–100% and blotting of NAD⁺ related proteins. Western blotting of FLAG confirmed the specific expression of the SLC25A51-construct. The dotted line indicates that the representative blots are from two different blots with their respective loading controls. (D) Quantification of WRN expression, confirming efficient knockdown of WRN in both HEK293 and SLC25A51. (E–G) Quantification of NAD⁺ related proteins. The groups were compared with Two-way ANOVA using Tukey’s multiple comparison to test the difference among all groups, and Student’s t-test as explained in Figure 2. NADSYN1 expression was increased in Scr cells with SLC25A51 overexpression compared to parental HEK293 cells (Two-way ANOVA, Tukey’s multiple comparisons test, p-value = 0.0048), and NAMPT was increased in both Scr and WRN-KD cells with SLC25A51 overexpression (Two-way ANOVA, Tukey’s multiple comparisons test, p-value < 0.0001 and p-value = 0.0004, respectively). No significant effects were found with 24 h 1 mM NR treatment.