Additional file 2 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 2: Supplementary Figure 2. [S2A] MAC681 treatment reduces the basal oxygen consumption rate in K-562 cells (double-sided unpaired t-test **p ≤ 0.01). [S2B] K-562 cells depend more on mitochondrial ATP than glycolytic ATP production. MAC681 treatment specifically reduces the mitochondrial ATP production rate (double-sided unpaired t-test **p ≤ 0.01). [S2C] Mitochondrial Ca2+ co-localization was confirmed by the calculation of Manders' coefficients (co-occurrence), Pearson's coefficient (correlation), and Spearman's coefficient (correlation). [S2D] Time-dependent translocation of AIF from mitochondria to the nucleus induced by MAC681 (5 µM). Immunofluorescence microscopy analysis of K-562 cells treated or not with MAC681, incubated with the AIF antibody, and counter-stained with Hoechst. Representative pictures from three independent experiments are shown. [S2E] Time-dependent translocation of AIF from the cytoplasm to the nucleus in K-562 cells, assessed by western blot. Tubulin and lamin B were used as loading controls. After quantifying the bands of interest, cytoplasmic and nuclear proteins were normalized to tubulin and lamin B, respectively (double-sided unpaired t-test *p ≤ 0.05, ****p ≤ 0.0001). Representative western blots from three independent experiments are shown. [S2F] MAC681-induced PARP1 degradation after 4 hours of treatment was prevented by calpain inhibitor PD 150606 (1-hour pre-treatment) in K-562 cells, assessed by western blot. β-Actin was used as a loading control. A representative picture from three independent experiments is shown. [S2G] Effect of MAC681 on leukemia cell line viability. The IC50 value was defined as the compound concentration needed to inhibit 50 % cell viability compared to untreated controls. [S2H] Differential toxicity of MAC681 in PBMCs and K-562 cells (left panel) or RPMI1788 and K-562 cells (right panel) was estimated based on results with trypan blue exclusion assay (One-way ANOVA, Šídák's multiple comparisons test ***p ≤ 0.001, ****p ≤ 0.0001). [S2I] Colony formation assay with K-562 cells treated with MAC681 at the indicated concentrations. Quantification of total MTT signal (One-way ANOVA, Dunnett’s multiple comparisons test ***p ≤ 0.001, ****p ≤ 0.0001). [S2J] Colony formation assay with U-937, Meg-01, KBM-5, and HL-60 cells treated with MAC681 at the indicated concentrations. Images are representative of three independent experiments. Neither viability nor proliferation of the leukemia cells was affected by MAC681 before seeding the pretreated cells on methylcellulose (right panels) (One-way ANOVA, Dunnett’s multiple comparisons test *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). [S2K] Effect of MAC681 on K-562 (upper panel) and U-937 (lower panel) tumor formation in zebrafish xenograft assays. Representative images from 10 fish per condition. PBS injection served as a negative control. Error bars represent the SD of at least three independent experiments. The fluorescent U-937 tumor mass was quantified and reported in the graph (One-way ANOVA, Dunnett’s multiple comparisons test *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).