SUPPLEMENTAL FIGURE LEGENDS
Supporting figure 1. Healthy cells are more resistant to HAMLET
AHealthy kidney cells (HRPTEC), kidney carcinoma cells (A498) and lung carcinoma cells (A549) were treated with HAMLET and ATP levels were measured after 3, 6 and 24 hours. At 21 μM HAMLET caused a reduction in ATP levels in the carcinoma cells but not in the healthy cells. At 36 μM healthy cells also showed a reduction in ATP but not to the same extent as the carcinoma cells. ATP levels in % of levels in respective untreated cells. Means of 3 experiments + SEMs.B Growth arrest following serum withdrawal (48 hours) and double thymidine block (2 x 16 hours) in A549 cells were confirmed by flow cytometry after staining with propidium iodide.
Supportingfigure 2. Glucose deprivation and the glycolytic inhibitors 2DG and 3-Bromopyruvate enhance HAMLET-induced cell death
Lung carcinoma cells were deprived of glucose (A-C) or treated with the glycolytic inhibitor 2DG (D, E) (30 minutes preincubation for both) and treated with HAMLET (21 and 36 μM, 1 and 3 hours). The cytotoxic effect of HAMLET was quantified by measuring ATP levels (shown in % of the level in untreated cells in 11.1 mM glucose) (A, D), Trypan blue (TB) exclusion (B, E) and release of LDH (HL, 21 μM, C). α-Lactalbumin (ALA, 21 μM) was used as control. Both glucose deprivation and 2DG enhanced the effect of HAMLET in a dose-dependent manner. Means of 3 (2DG) or 4 (glucose deprivation) experiments + SEMs or results from 1 experiment (LDH release). ** for p ≤ 0.01, *** for p ≤ 0.001 (vs. untreated, 11.1 mM glucose for glucose deprivation and vs. untreated, no 2DG for 2DG). (F) 3-Bromopyruvate increased the effect of HAMLET in a dose-dependent manner and even had a cytotoxic effect by itself. ATP levels in % of levels in untreated cells. Means of 4 experiments + SEMs. * for p ≤ 0.05, ** for p ≤ 0.01, *** for p ≤ 0.001 (vs. respective HL, no 3-Bromopyruvate).
Supportingfigure 3. HAMLET shows only weak colocalization with PKM
HAMLET showed weak colocalization with PKM especially in the perinuclear region. A549 cells were treated with Alexa Fluor 568-labeled HAMLET (HL, red) for 15 minutes and stained with anti-PKM and Alexa Fluor 488-labeled antibodies (green). Size bars: 20 µM.
Supporting figure 4. HAMLET’s effect on glycolytic enzymes, ATP and lactate levels
AA549 cells were treated with different HAMLET concentrations for 15 minutes, 1 and 3 hours and examined for morphological changes. After 15 minutes no changes in cell morphology were observed but after 1 hour cells showed blebbing and contracted and after 3 hours the majority of cells had rounded up completely. B HK1 and PKM2 protein levels in HAMLET-treated A549 cells (15 minutes) were examined by Western blot. No changes were observed after HAMLET treatment. CIn A549 cells, HAMLET reduced ATP after 15 minutes and lactate levels in the medium after 3 hours. ATP levels in % of levels in untreated cells at respective time points. Means of 3 (lactate) or 4 (ATP) experiments ± SEMs.
Supporting figure 5. Hierarchical clustering based on significantly altered features in the reverse phase LC-MS analysis
A Cells were treated with HAMLET for 15 minutes or 1 hour and metabolites were analyzed by LC-MS reverse phase chromatography. Hierarchical clustering was performed based on significantly altered features (abs. log2 fold change 1and p value ≤ 0.05) for each time point.
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