Additional file 3 of Antileukemic potential of methylated indolequinone MAC681 through immunogenic necroptosis and PARP1 degradation
Creators: name: Orlikova-Boyer, Barbora
name: Lorant, Anne
name: Gajulapalli, Sruthi Reddy
name: Cerella, Claudia
name: Schnekenburger, Michael
name: Lee, Jin-Young
name: Paik, Ji Yeon
name: Lee, Yejin
name: Siegel, David
name: Ross, David
name: Han, Byung Woo
name: Nguyen, Thi Kim Yen
name: Christov, Christo
name: Kang, Hyoung Jin
name: Dicato, Mario
name: Diederich, Marc
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Hematology

description: Additional file 3: Supplementary Figure 3. [S3A] Kinetic analysis of the PARP1 protein levels in K-562 cells treated with MAC681 at 5 µM. β-actin was used as a loading control. Etoposide (VP16) and ethanol 10% (Et10%) were used as positive controls. [S3B] RT-PCR analysis of PARP1 mRNA expression at indicated time points. K-562 cells were treated with MAC681 (5 µM), 3ABA (5 mM), or their combination (One-way ANOVA, Šídák's multiple comparisons test *p ≤ 0.05, **p ≤ 0.01). [S3C] Kinetic analysis of the PARP1 protein levels in K-562 cells treated with MAC681 at 5 µM with or without cycloheximide (CHX). β-actin was used as a loading control. Mcl-1 protein expression with short turnover was used as a positive control for CHX activity. [S3D] The control docking experiment of PARP1 with 2, 3-dihydrobenzofuran-7-carboximade derivative (2US). The 2US in the complex structure of PARP1 (PDB ID: 4OQA) and the docking model are shown as red and blue stick models, respectively. Oxygen atoms are visualized in orange (upper panel). MAC681 has a similar binding mode to 2US in a complex structure of PARP1 (PDB ID: 4OQA) and PJ34 in a complex structure of PARP1 (PDB ID: 4UXB). MAC681, 2US, and PJ34 are represented as stick models in magenta, red, and yellow, respectively (lower panel). PatchDock server and AutoDock4 program were used in our docking simulation studies. [S3E] The formation of γH2AX nuclear foci in U-937, MOLM-13, MV4-11, and OCI-AML3 was quantified by flow cytometry after 4 h of MAC681 treatment (left panel) (One-way ANOVA, Dunnett’s multiple comparisons test **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). Nuclear morphology was assessed by fluorescent microscopy with Hoechst staining at the same time point (right panel). [S3F] Percentage of cell death was quantified with Hoechst/PI staining in U-937 cells treated either by MAC681 (5 µM), 3ABA (1mM), VP16 (100 µM) or in a combination of 3ABA with MAC681 or with VP16 at 24 hours (upper panel). Western blot analysis of PARP1 protein cleavage in U-937 cells was determined simultaneously (lower panel). Etoposide (VP16) served as a positive control for apoptosis induction and PARP1 cleavage. [S3G] 3ABA-MAC681 adduct formation in K-652 cells (duplicate experiment). The 3ABA-MAC681-6-position adduct was detected by mass spectrometry in K-652 cells. Experimental conditions are described in the Materials and Methods section, Mass Spectrometry. MAC681-alcohol and trifluorophenol (TFP) are metabolites of MAC681. [S3H] Effect of washout of 3ABA pre-treatment on the protective effect against MAC681-mediated early and late cellular responses. Intracellular ATP content in K-562 cells pre-treated with 3ABA (5 mM) for 30 min, with or without washout, and treated for 4 hours with MAC681 (5µM) normalized to control cells was measured by CellTiter-Glo (upper left panel). Number of viable cells (upper middle panel) and viability (upper right panel) were assessed by trypan blue exclusion test after 24 h (two-way ANOVA, Fisher’s test *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). The nuclear morphology of cells was visualized using Hoechst/PI staining by fluorescent microscopy (lower panel) at 24 h. [S3I] Kinetic analysis of reactive oxygen species (ROS) levels measured by flow cytometry after MAC681 (5 µM) treatment with dichlorofluorescein diacetate (DCFDA) staining (upper panel) or with redox sensor red (RSR) staining (lower panel). H2O2 was used as a positive control for ROS induction (double-sided unpaired t-test *p ≤ 0.05, ****p ≤ 0.0001). [S3J] 2 e- reductase-dependent bioreductive mechanism of MAC681 activation via the iminium salt. [S3K] 2 e- reductase-dependent bioreductive mechanism of MAC681 activation via the quinone methide. [S3L] NQO1 (upper panel) and NQO2 (lower panel) expression levels in CML patient stem cells compared to stem cells of healthy volunteers in indicated datasets (GSE5550, GSE97562+ GSE47927) (double-sided unpaired t-test *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). [S3M] NQO1 (upper panel) and NQO2 (lower panel) gene expression profiles in different CML cancer cell lines from the CCLE dataset.