Fig. 1

Pharmacological and genetic inhibition of PARP1 and MGMT potentiates temozolomide cytotoxicity in a linear fashion and is associated with PARP1-MGMT interaction. a TMZ-treated (0–3 mM) Ewing sarcoma cell lines exposed to TLZ (IC₁₀) and O⁶BG (5 μM) for 96 h (Alamar Blue assay). EW-8 cell line is shown as a model example, additional results for ES-4, ES-6, and ES-7 cell lines are available in Additional File 1: Fig. S1a. TLZ, talazoparib. TMZ, temozolomide. O⁶BG, O⁶-benzylguanine. b Potentiation to TMZ: IC₅₀ values for EW-8 cell line as in a. EW-8 cells are intrinsically resistant to TLZ [8]. P-values are calculated for TMZ vs TMZ + TLZ, TMZ + O⁶BG, TMZ + TLZ + O⁶BG by ANOVA3 followed by Tukey’s test for multiple comparisons: ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05. Legend colors are coordinated with colors in a. c MGMT and d PARP1 gene knockdown-induced potentiation to TMZ (IC₅₀, 48 h) in ES-7 and EW-8 cells (RNAi high-throughput screen). Readout is ATPlite cell viability assay. Each bar represents mean IC₅₀; error bars are calculated for 3 siRNAs run in triplicate. P-values calculated by t-test, non-paired, un-equal variance, 2-sided: ****p ≤ 0.0001; ***p ≤ 0.001. PARP1 gene knockdown was not as effective as talazoparib, which inhibits PARP1 and PARP2 (the latter is linked to toxicity [9]). e TMZ treatment of EW-8 cells (0–1 mM) ± MGMT or g PARP1 gene knockdown by siRNA (Alamar Blue staining). Student’s paired 2-tailed t-test: p = 0.05 (e); *p ≤ 0.05 (g). f MGMT or h PARP1 protein downregulation by siRNA (Western blot at 48, 72, 96 h). GAPDH (37 kDa). Beta-actin (43 kDa). NT, no treatment. i EW-8 cells ± TMZ treatment (1 mM, 2 h): PARP1 pulldown was followed by PARP1 (top) or MGMT (bottom) immunoblotting. Lanes 1–2: co-immunoprecipitation. Lane 3: IgG1. Lanes 4–5: input. j Mean of protein band intensities generated from 3 independent co-immunoprecipitation experiments in (i). Student’s paired 2-tailed t-test: *p ≤ 0.05; **p ≤ 0.01 (see reverse co-IP in Additional File 1: Fig. S2c, d). k Negative (PARP1-GAPDH) and positive (PARP1-PARP2) interactions by co-immunoprecipitation in EW-8 cells. Samples prepared as in (i). IgG control is in middle lane. l Representative image of EW-8 cells nuclei staining with Hoechst 33,342 (blue, nuclei), Alexa Fluor 647 (red, MGMT), and Alexa Fluor 488 (green, PARP1). White pixels indicate green and red overlap, i.e., co-localization of PARP1 and MGMT. Top panel, no TMZ. Bottom panel, TMZ at 1 mM for 2 h. m Scatterplot representing red (MGMT) and green (PARP1) pixel intensities in (l); overlap of these colors along the diagonal in the field ‘c’ (~ 45°) corresponds to protein co-localization dots (shown as white pixels). Co-localization analysis was done using CellSens software (v2.1). Images were developed with Fluoview FV3000. n Quantification of white-pixel number of co-localized PARP1-MGMT sites in control vs TMZ-treated EW-8 cell nuclei. Data from 3 independent experiments were used for the analysis. NT, no treatment. o SDS-PAGE and silver staining of protein gel showing PARP1-MGMT interaction by immunoprecipitation assay. Pulldown with full-length PARP1. p Purified PARP1 and MGMT protein interaction. Mixed full-length PARP1 and MGMT proteins (1:1) were subjected to co-immunoprecipitation. PARP1 was pulled down with the co-immunoprecipitation specific PARP1 antibody (cst-9532) and immunoblotted with PARP1 (top; cst-9542) or MGMT (bottom; sc-241154) antibodies. IgG control is in the middle lane. q Purified N-terminal of PARP1 (aa 1–662) was mixed with full-length MGMT (1:1) and processed for co-immunoprecipitation. Samples prepared as in (p). IgG control shown in middle lane. r An MST-on time of 10 s analysis of the full-length PARP1 and MGMT protein affinity was performed using Monolith NT.115 at 17% LED power and medium MST power