Figure 4.

Lack of NMNAT3 and the NMN Transporter SLC12A8 Extends Nuclear ADP-Ribosylation after H₂O₂ Treatment

(A) Schematic overview of compartmentalized NAD⁺ synthesis and breakdown in most transformed cells.

(B and C) U2OS cells were transfected with siRNA targeting SLC12A8, and nuclear (left) or mitochondrial (right) ADP-ribosylation was analyzed via IF and quantified at various time points following H₂O₂ treatment.

(D) NMNAT3 was knocked down in U2OS, and nuclear (left) or mitochondrial (right) ADP-ribosylation was analyzed via IF using an anti-ADPr antibody.

(E) U2OS cells were pre-treated for 1 h with rotenone or oligomycin prior to a 10-min treatment with H₂O₂ and nuclear and mitochondrial ADP-ribosylation was analyzed via IF.

(F) Mitochondrial NAD⁺ levels following rotenone treatment were assessed in stable U2OS Flp-In T-Rex cells expressing an inducible mitochondrial NAD⁺ sensor and analyzed via flow cytometry. Increased FRET ratios correspond to increased NAD⁺ levels. FRET ratios of three independent experiments including square deviation are shown.

(G) IF using the anti-ADPr antibody of U2OS cells pre-treated with rotenone and subsequently treated for 3 h with MMS. The quantifications of all signals (mitochondrial and nuclear) were normalized as described in Figure 1, and the y axes of all plots are depicted as log10 scale. Scale bars indicate 20 μm. For statistical analysis, a Student’s t test was performed (n = 3–5; ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005).