Medjkane et al. ONC-2012-02287R - Supplementary information

Theileria Induces Oxidative Stress and HIF1α Activation

that are Essential for Host Leukocyte Transformation

Souhila Medjkane 1, Martine Perichon 1, Justine Marsolier 1,

Julien Dairou 2 & Jonathan B. Weitzman 1‡

Supplementary information

Supplementary Information Figure S1 : Theileria induced a Warburg-like effect in the TBL20 cell line

Quantitative PCR (qPCR) analysis of the expression level of Hexokinase 2 (HK2), lactate dehydrogenase (LDHA), pyruvate dehydrogenase kinase (PDK1) and the glucose transporter GLUT1 using RNA from Theileria infected TBL20 cells or BL20 non-infected cells, with/without Buparvaquone treatment (Bup) for 64 hrs. β-actin mRNA was used for normalization. (average ± sd, n=4).

Supplementary Information Figure S2 : The Warburg effect was reversible upon Theileria elimination in H7 and Thei macrophage cells

A/B- Quantitative PCR (qPCR) analysis of the expression level of HK2, LDHA, PDK1 and GLUT1 using RNA from Theileria-infected H7 and Thei cells, with/without Buparvaquone treatment (Bup) for 64 hrs. β-actin mRNA was used for normalization. (average ± sd, n=3).

C/D- Elimination of Theileria by Buparvaquone (Bup) treatment for 64 hrs led to a decreased HIF1α protein as assessed by Western analysis in two independent macrophage cell lines : H7 and Thei. β-actin was used as a loading control.

Supplemental information Figure S3 : HIF1α is required for the transformation induced by Theileria in H7 macrophage cell line.

A- Western-blot analysis of HIF1α expression in Theileria-infected H7 macrophage cells treated for 24hr with 40 nM Digoxin or 20 nM Ouabain. β-actin was used as loading control.

B- Parasite-infected H7 cells formed colonies when grown in soft agar. This transformed phenotype was reversed by incubation with the inhibitors of HIF1α : Digoxin at 40 nM (Dig 40) or Ouabain at 20 nM (Ouab 20). The relative number of colonies per plate and their appearance under the microscope 10 days after plating are shown. (average ± sd, n=2) **p<0.01; ***p<0.001 .

Supplementary Information Figure S4 : The effects of drug treatment on apoptosis, signaling pathways and ROS production.

A- Apoptotic cells (Annexin V-positive and propidium iodide PI-negative cells) were monitored in parasite-infected TBL3 cells treated with the inhibitors of HIF1α : Digoxin at 20 and 40 nM (Dig 20, Dig 40) or Ouabain 10 nM and 20 nM (Ouab 10, Ouab 20) or silenced for HIF1a expression using two independent siRNA oligonucleotdes. TBL3 cells were also treated with the antioxidant NAC 4 mM for 24hrs. Etoposide treatment was used as a positive control for the apoptosis assay.

B- Western-blot analysis of c-Myc, c-Jun and phospho-c-Jun expression (as indicated by the asterisks) in infected TBL3 cells treated for 24 hrs with NAC at 4 mM. β-actin was used as a loading control.

C- Luciferase assays showing the expression of an (-) empty vector or (+) AP1-luciferase construct or NF-κB-luciferase construct transfected into TBL3 cells and treated (+) or not (-) with NAC 4 mM. Transfection efficiency was normalized with co-transfection of a Renilla-encoding plasmid. (average ± sd, n=2).

D- Representative flow plot for ROS production as measured by dihydroethidium staining (DHE) detected by flow cytometry in FL3 channel for TBL3 or BL3 cells treated with Bup or the indicated HIF1α inhibitors.

Supplemental information

Materials and Methods

GSH:GSSG ratio measurement

The procedures for the measurement of GSH and GSSG were designed to minimize artificial oxidation of GSH to GSSG. GSH and GSSG were separated by HPLC, equipped with a Shimadzu Prominence solvent delivery system (Shimadzu Corp., Kyoto, Japan), using a reverse-phase C18 Kromasil (5 μm; 4.6 × 250 mm), obtained from AIT (Paris, France). The mobile phase for isocratic elution consisted of 25 mmol/L monobasic sodium phosphate, 0.3 mmol/L of the ion-pairing agent 1-octane sulfonic acid, 1% (v/v) acetonitrile, pH 2.7, adjusted with 85% phosphoric acid. The flow rate was 1 ml/min. Under these conditions, the separation of aminothiols was completed in 20 min; GSSG was the last eluting peak, with a retention time of 15 min. Deproteinated samples were injected directly onto the column using a Shimadzu autosampler (Shimadzu Corp.). Following HPLC separation, aminothiols were detected with a model 2465 electrochemical detector (Waters) equipped with a 2 mm Glassy carbon (GC) analytical cell and potential of +875 mV were applied . For the cells in standard conditions, we obtained one molecule of GSSG for 25-35 molecules of GSH.

Production of ROS metabolites as measured by flow cytometry using dihydroethidium

Dihydroethidium was used for the flow cytometry measurement of superoxide production. Dihydroethidium is rapidly oxidized to ethidium (red fluorescent compound) by H2O2 (in the presence of peroxidase) and superoxide. The cells were stained with dihydroethidium (2 μmol/l) by incubating for 30 min at room temperature in the dark and analysed in a FACSCalibur flow cytometer (Becton Dickinson, San Juan, U.S.A.). Fluorescence was measured using filter FL-3. Histograms of 20 000 events per experiment were analysed. Cells with dihydroethidium fluorescence were then evaluated by using the Cell Quest software.

Measurement of Lactate production

Samples for lactic acid production were analyzed through HPLC with UV detector (Shimadzu Prominence LC20) at 214 nm using RP-18 column (250 x 4.6 mm). The operating conditions were: isocratic condition with mobile phase KH2PO4 (20 mM) adjusted to pH=2.8; flow rate 0.8 mL/min; 40°C column temperature.

Measurement of LDHA activity

Lactate dehydrogenase (LDHA) is an oxidoreductase enzyme that catalyses the conversion of pyruvate to lactate. The measurement of LDHA activity was based on the rate of decrease in the absorbance at 340 nm, resulting from the oxidation of NADH. LDHA activity was detected in a total volume of 200 µl. Samples containing crude cells extract were first incubated with pyruvate (1 mM final concentration) in PBS at 37°C for 5 min. NADH (1 mM final concentration) was added to start the reaction, the samples were incubated for various periods (up to 30 min) at 37 °C and the absorbance was measured at 340 nm. All assays were performed in triplicate under initial reaction rate. After washing with PBS buffer, cells were

analysed by flow cytometry.

Measurement of Glucose uptake

Cells were incubated 20 min at 37ºC in media supplemented with the fluorescent D-glucose analog 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose), (Life Technologies; N13195) at 100 µM for 20 min at 37ºC. After washing with PBS, cells were analyzed by flow cytometry, fluorescence assessed in FL1 channel. For each measurement, data from 10000 single cell events were collected using a FACScalibur flow cytometer (Becton Dickinson Immunocytometry Systems, SanJose, CA).

Cell cycle and Apoptosis analysis

For S-phase analysis, cells fixed in 70% cold ethanol and washed twice in PBS, were incubated for 30 minutes at room temperature in PI buffer (50 μg/ml propidium iodide (PI); Sigma) containing 100 μg/ml RNaseA. Cells were analyzed using the FACScalibur flow cytometer, data from 10000 single cell events were collected using a FACScalibur flow cytometer and data analysed using CellQuest software (Becton Dickinson Immunocytometry Systems, SanJose, CA).

For Annexin/PI staining, samples were resuspended in 0.5 ml calcium-containing Annexin binding buffer (Pharmingen). Annexin-FITC (5 μl) was added and samples incubated for 15 minutes at room temperature in the dark. 1 μl of 100 μg/ml PI were added immediately before running on the FACSCalibur. Annexin-FITC fluorescence was measured using a 530 nm bandpass filter and PI fluorescence using a 670 nm longpass filter. At least 10,000 events were acquired from each sample. Events were scored as apoptotic if they were annexin-positive and PI-negative.