Additional file 1 of The troglitazone derivative EP13 disrupts energy metabolism through respiratory chain complex I inhibition in breast cancer cells and potentiates the antiproliferative effect of glycolysis inhibitors
Creators: name: Muller, Claire
name: Lacroix-Malgras, Victorine
name: Kluza, Jérôme
name: Laine, William
name: Güler, Yonca
name: Bost, Frédéric
name: Boisbrun, Michel
name: Mazerbourg, Sabine
name: Flament, Stéphane
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Cancer
subject: Hematology

description: Additional file 1: Figure S1.. EP13 and 2-DG act synergistically to kill breast cancer cells. MDA-MB-231, Hs578T, and MCF-7 cells were treated for 72 h with increasing concentrations of EP13 and 2-DG used alone or in combination at a constant ratio of 1/1. In this last condition, the total dose corresponds to the addition of the concentration value of EP13 and 2-DG used in combination (a total dose of 2 corresponds to treatment with 1 µM EP13 and 1 mM 2-DG). Cell numbers were determined by crystal violet staining. The results are depicted as the mean ± SEM of at least three independent measurements. Significant differences from control cells are indicated. *, p &lt; 0.5; **, p &lt; 0.01; ***, p &lt; 0.001. Figure S2. EP13 inhibits mitochondrial respiration after 24 h treatment. MDA-MB-231 (a) and MCF-7 (b) cells were treated for 24 h with vehicle or EP13 (6 μM), and the OCR was measured using an XFe24 Seahorse system. At the times indicated, the following drugs were injected: oligomycin A (Oligo; 2 µM), FCCP (1.1 µM and 2.2 µM), and rotenone / antimycin A (Rot/AA; 1 µM each). Data are the means ± S.D.s (at least n = 3 wells per group). Figure S3. Methyl pyruvate does not reverse the EP13 effect in breast cancer cells. MDA-MB-231 (a) and MCF-7 (b) cells were treated for 48 h with EP13 (6 µM) and methyl pyruvate (5 mM). Control cells were exposed to vehicle. Cell numbers were determined by crystal violet staining. The results are depicted as the mean ± SEM of at least three independent measurements. Significant differences from control cells are indicated. ***, p &lt; 0.001; ns, not significant. Figure S4. EP13 increases glucose uptake in MDA-MB-231 cells. MDA-MB-231 cells were treated for 4 h with EP13 (3 and 6 µM) or vehicle (DMSO). Glucose uptake was assessed by incorporation of the fluorescent glucose analog 2-NBDG for 1 h in glucose-free DMEM. The median fluorescence intensity was measured and normalized to unstained controls. The protocol is described in Additional file 2. The results are depicted as the mean ± SEM of at least three independent measurements. Significant differences from control cells are indicated. **, p &lt; 0.01. Figure S5. EP13 and dichloroacetate (DCA) show additive effects to kill breast cancer cells. MDA-MB-231 cells were treated for 72 h with EP13 (3 and 6 µM) and increasing concentrations of DCA, an inhibitor of pyruvate dehydrogenase kinase (PDK), used alone or in combination. Cell numbers were determined by crystal violet staining (a). The results are depicted as the mean ± SEM of at least three independent measurements. Two-way analysis of variance (ANOVA) followed by the Dunnett multiple comparisons posttest were performed. Significant differences from control cells are indicated. **, p &lt; 0.01; ***, p &lt; 0.001. Data were analysed by Compusyn software to determine the combination index (CI) with a nonconstant ratio according to the Chou Talalay method (b). The area between the dashed line (0.9 &lt; CI &lt; 1.1) is related to an additive effect. A CI &gt; 1.1 indicates an antagonistic effect, and a CI &lt; 0.9 indicates a synegistic effect. The protocol is described in Additional file 2.