Fig. 2

Alkylating DNA damage intensifies O⁶meG repair through PARylation of MGMT. a SDS-PAGE/Western blot (top and middle) and BSA Ponceau staining (bottom) for PAR and MGMT. Key: Ss/dsOligo1 is MCAT; ss/dsOligo2 is MGMT-Oligo; ss/dsOligo3 is ss/dsMGMT-O⁶meG. The resulting proteins were detected by SDS-PAGE analysis followed by Western blot for PAR (top) and MGMT (bottom). See PARP1 Western blot in Additional File 1: Fig. S3b. BSA, Ponceau S membrane staining. PAR-PARP1 is auto-PARylated PARP1. PAR-MGMT is PARylated MGMT. b Glutathione-S-transferase (GST) is a non-binding substrate of PARP1 and is not PARylated (serves as control). The purified PARP1, GST proteins, NAD⁺, and dsOligo1 were processed as in (a). c ELISA assay to evaluate PAR levels in Ewing sarcoma EW-8, rhabdomyosarcoma (RD), and fibroblast HFF1 cell lines ± temozolomide treatment (1 mM, 2 h) using SpectraMax M5 plate reader (450 nm). Student’s paired 2-tailed t-test: **p ≤ 0.01. NT, no treatment. TMZ, temozolomide. d MGMT repair assay diagram. The repair product is cleavable by PvuII restriction digestion. The unrepaired O⁶meG dsDNA (intact, i.) and repair product (cleaved, c.) can be analyzed by gel electrophoresis. e MGMT repair assay. MGMT and PARP1 (6.2 nM) were incubated with MCAT dsDNA for 1 h at 37°C to induce PARylation and then incubated with ³²P-labeled-O⁶meG-dsDNA (50 nM) for repair reaction. Reaction products were analyzed by PvuII treatment followed by PAGE and phosphor-imaging. f % of repair results quantified using Image J as a ratio of cleaved band intensity to a sum of intact and cleaved band intensities from (e) were plotted (by Prism 8). Stronger increase in DNA cleavage (O⁶meG repair) was observed in the presence of PARP1 and NAD⁺. Student’s paired 2-tailed t-test: **p ≤ 0.01. g PARylation activity in EW-8 cells in response to short- (2 mM, 2 h) and long-term (100 μM, 72 h) temozolomide treatment by Western blot for PAR, PARP1, MGMT, and GAPDH proteins. PAR-PARP1 is PARylated PARP1. h Quantified band intensities for PAR, PARP1, and MGMT bands normalized to GAPDH levels (n = 3) and plotted using Image J. GAPDH (37 kDa) is loading control. i Chromatin and nuclear soluble fractions of EW-8 cells treated with temozolomide at 2 mM for 2 h or at 100 μM for 72 h by Western blot. Histone 3 (15 kDa) is chromatin fraction control. SP1 (81 kDa) is nuclear soluble fraction control