Figure 3. Absence of PAR or PAR-catabolite mediated cell death following BER failure.

(A) Model depicting the nexus of BER, the synthesis of PAR and the generation of PAR catabolites in response to BER failure-induced PARP1/PARP2 hyperactivation.

(B) Absence of mitochondria to nucleus translocation of AIF due to BER failure as determined by confocal microscopy. BER deficient cells (LN428/MPG) were treated with media (left panel) or 1.5 mM MMS (right panel) for 1 hour and then washed and allowed to recover in media for 5 hours prior to fixation and staining for AIF (green), actin (red) and nucleus (blue).

(C) PARG KD prevented degradation of DNA damage-induced PAR. (Left Panel) Immunoblot of PAR to determine the degradation of PAR in LN428/MPG/PARG-KD cells following treatment with 1.5 mM TMZ. PCNA protein expression level was shown as a loading control. (Right Panel) Preventing generation of PAR catabolites from degradation of PAR via PARG KD enhances TMZ-induced cytotoxicity. LN428 and LN428/MPG cells with (black solid bars) or without (white empty bars) PARG KD were exposed to TMZ (1 mM) or vehicle control (DMSO) for 48 hours. Viable cells were determined as in Figure 1B and reported as percentage relative to vehicle control treated cells (% control). Results indicate the mean ± S.E. of three independent experiments.

(D) HMGB1 released into the cell culture media, as demonstrated by immunoblot. LN428 and LN428/MPG cells were pre-treated with PJ34 (4 μM) or vehicle control for 30 min and then exposed to TMZ (1.5 mM) with or without PJ34 (2 μM) for 12 hours. HMGB1 was then captured using immobilized heparin and analyzed by immunoblot, as described in the Materials and Methods section.