Intensity % Intensity % A Human recombinat MIF protein (hrmif), MW: 12428.31 Da m/z hrmif (12428.31 Da) + 4-IPP (282 Da) MWtot ~ 12715.21 Da m/z B HTC/C3 DAPI phistone-h3 Merge HTC/C3 DAPI phistone-h3 Merge UNT 4-IPP 5 µm 72h DMSO 4-IPP 10 µm 72h 4-IPP 2,5 µm 72h 4-IPP 25 µm 72h C TPC-1 ALEXA-MIF, DAPI CD74, DAPI ALEXA-MIF, CD74 D TPC-1 4-IPP [μm] 24h p53 Tubulin
E Ebselen treatment [24h] of TPC-1 cell line Unt. 24h DMSO 24h Ebselen 5 µm 24h Ebselen 10 µm 24h Ebselen 25 µm 24h Ebselen 50 µm 24h F TPC-1 Lysates [24h] MIF perk1/2 (T185/Y183) Cyclin-D1 Cyclin-B1 Tubulin
G FACS analysis (TPC-1 cell line, untreated control group) Untreated 0h Untreated 12h Untreated 24h Untreated 48h Untreated 72h
H FACS analysis (K1 cell line, untreated control group) Untreated0h Untreated 12h Untreated 24h Untreated 48h Untreated 72h
I Glucose Hexokinase Glucose-6P Phosphoglucose isomerase Fructose-4P Phosphofrutto Kinase Fructose-1,6 bip Mitochondrion Glyceraldheyde-3-P Dihydroxyacetone-P NAD Pyruvate PDH AcetylCoA NADHH + TCA cycle OXPHOS Phosphoenolpyruvate Citrate Pyruvate Kinase NADH+H+ Pyruvate Dehydrogenase Pyruvate NAD Citrate + CoA TCA cycle Lactate -citrate lyase Oxaloacetate + CO 2 Acetil-CoA Malonyl-CoA Acetil-CoA Carboxylase Cholesterol Synthesis Palmitate Figure legend Hexokinase: Enzyme TCA: Trycarboxilic Acid Cycle P: Phosphate OXPHOS: Oxidative phosphorylation PHD: Pyruvatedehydrogenase CoA: Coenzyme A
SUPPLEMENTARY FILE 1. (A) MALDI-TOF mass spectrometry was carried out on Voyager-DE STR mass spectrometer (Applied Biosystem, Framingham, MA, USA). Peptide solution were mixed 1:1 with a saturated solution of sinapinic acid in 33% acetonitrile and 0,1% trifluoroacetic and allowed to air dry on the sample plate before the analysis. hrmif was preincubated in a presence of excess (10-fold) of 4-IPP for 3h, therefore hrmif was analyzed by MALDI-TOF mass spectrometry in the linear mode. The calculated molecular weight (M/W) of hrmif was 12428.31 Da, 4-IPP was 282 Da and rmif + 4-IPP was 12716.21. After incubating rmif and 4-IPP for 3 h at room temperature, we observed a mass shift of approximately 282 Da, which corresponded to the molecular mass of 4-IPP; thus, 4-IPP could effectively recognize its target. The figures show data from a representative experiment. All the experiments were performed in triplicate. (B) 4-IPP inhibitor leads to G2/M block through the increase of histone H3 phosphorylation in CD74-positive anaplastic thyroid carcinoma cell line. Coverslips with HTC-C3 cells were incubated with 2,5; 5; 10 and 25 μm 4-IPP or with DMSO (vehicle) for 72 h. Then cells were fixed and stained with anti-phistone H3 (S10) antibody (red) to visualize histone H3 and with DAPI (blue) to visualize cell nuclei. Fluorescence micrographs show untreated HTC-C3 cells with a weak/moderate phosphorylation signal of histone H3. After 72 h of 4-IPP treatment, phosphorylation signal of histone H3 was increased in HTC-C3 cells. These results suggest that 4-IPP treatment should lead to G2/M cell cycle block. The figures show representative data from a representative experiment. All immunofluorescence experiments were performed in triplicate. (C) 4-IPP treatment impaired CD74/CD44 complex mediated MIF internalization. Immunostaining of TPC-1 cells treated with recombinant Alexa 546 MIF inactivated with 4-IPP. Alexa 546 MIF was incubated in the presence of an excess (10-fold) of 4-IPP overnight prior to being added to the culture medium, and subsequently TPC-1 cells were fixed after 30 min of incubation. Magnification: 60x Scale bar: 50 μm. The figures show representative data from a representative experiment. All immunofluorescence experiments were performed in triplicate. (D) Western blot analysis of p53 expression in TPC-1 cell line treated with 4-IPP at 10, 25, 50 and 100 μm after 24h. Normalization was obtained with immunoblot analysis of β-tubulin. Western blot analysis were performed in triplicate. (E) Micrographs of TPC-1 cells before and after drug treatments. Ebselen treatment induced a marked morphological change in TPC-1 cells, resulting in a smaller, contracted appearance and decreased cell spreading. Magnification: 10x. Scale bar: 500 µm. The figures show data from a representative experiment. All the experiments were performed in triplicate. (F) Western blot analysis of MIF, perk-1/2, Cyclin-D1 and Cyclin-B1 in TPC-1 cell line with Ebselen Selenoxide at 5, 10 25, 50 µm after 24h. Normalization was obtained with immunoblot analysis of β-tubulin. Western blot analysis were performed in triplicate.
(G) Cell cycle analysis of TPC-1 untreated cells. Representative flow cytometric profile of cell cycles in untreated TPC-1 cells, analyzed with propidium iodide staining. Analysis was performed with the CellQuest program (Becton Dickinson). The figure show data from a representative experiment. Treatments and the percentage of cells in the different phases of cell cycle are indicated. All the experiment were performed in triplicate. (H) Cell cycle analysis of K1 untreated cells. Representative flow cytometric profile of cell cycles in K1 untreated cells, analyzed with propidium iodide staining. Analysis was performed with the CellQuest program (Becton Dickinson). The figure show data from a representative experiment. Treatments and the percentage of cells in the different phases of cell cycle are indicated. All the experiments were performed in triplicate. (I) Flow Chart: Glucose enters the cytosol, and is oxidated in the glycolysis pathway, which consumes 2 to form glucose 6-phospate and fructose 1,6 bisphosphate. Aldolase breaks the glucose molecule to form two glyceraldeyde 3-phosphates. After several steps, pyruvate is produced with 2 and 2 NADHH molecules. In aerobic metabolism, pyruvate is transferred to the pyruvate-dehydrogenase complex in the mitochondrial matrix, and the dicarbon units are metabolized in the trycarboxilic acid (TCA) cycle, followed by oxidative phosphorylation (OXPHOS). Citrate can leave the mitochondrial inner space, and it is cleaved by -citrate lyase into Acetyl CoA, which is the dicarboxilic unit for cytosolic synthesis of fatty acids and cholesterol. Malic enzyme generates a new pyruvic acid, which can return to the mitochondria for metabolism. In the presence of inefficient OXPHOS and TCA activities, it is likely that pyruvate dehydrogenase cannot deliver all the pyruvate into the mitochondria. To maintain sufficient energy, glycolysis is enhanced, and pyruvate is converted into lactate by lactic dehydrogenase. Data values represent the average of three independent experiments.