Figure 4. BER failure induced cell death depends on NAD⁺ availability.

(A) Alkylation damage promotes NAD⁺ and ATP depletion in BER defective cells. NAD⁺ content (left panel): Cells were treated with media (white bars) or 0.5 mM MMS (black bars) for 1 hour prior to collection for NAD⁺ content analysis via enzymatic assay as described in the Materials and Methods section. ATP content (right panel): Cells were treated with media (white bars), 0.5 mM MMS (grey bars) or 1.5 mM MMS (black bars) for one hour. ATP content was measured after 1-hour recovery in normal media via the luminescence ATP assay described in the Materials and Methods section. NAD⁺ levels or ATP levels shown are the average of three independent experiments and are reported as percent control of the untreated control cell line.

(B) PARG-KD does not rescue alkylation damage induced NAD⁺ and ATP depletion in BER defective cells. NAD⁺ content (left panel): PARG-KD cell lines were treated with media (white bars) or 0.5 TMZ (black bars) for one hour prior to collection for NAD⁺ content analysis as described in the Materials and Methods section. ATP content (right panel): PARG-KD cells were treated with media (white bars), 0.5 mM TMZ (dotted bars), 1.0 mM TMZ (grey bars) or 1.5 mM TMZ (black bars) for one hour. ATP content was measured after 1-hour recovery in normal media via the luminescence ATP assay described in the Materials and Methods section. NAD⁺ levels or ATP levels shown are the average of three independent experiments and are reported as percent control of the untreated control cell line.

(C) Bioenergetic metabolites rescue Pol ß deficient cells from DNA damage-induced cell death. LN428 and LN428/MPG cells were pre-treated with NMN, NA or vehicle control (media) for 24 hours and were then exposed to TMZ (1 mM) in the presence or absence of NMN or NA for 48 hours. Viable cells were measured as in Figure 1B and were reported as percentage relative to vehicle control treated cells (% control). Results indicate the mean ± S.E. of three independent experiments.

(D) NAD⁺ biosynthesis inhibition augments BER failure-induced cell death. Cells were pretreated for 24 hours with a non-toxic 10nM dose of FK-866 (black bars) or DMSO (white bars). Cells were then exposed to media control or MMS (0.5 mM) for 1 hour. Viable cells were determined as in Figure 1B and results indicate the mean ± S.E. of three independent experiments.