HeLa cells stably transfected with an empty pcdna3 vector (HeLa Neo) or with a plasmid

Transcription

1 SUPPLEMENTAL MATERIALS AND METHODS Cell culture, transfection and treatments. HeLa cells stably transfected with an empty pcdna3 vector (HeLa Neo) or with a plasmid encoding vmia (HeLa vmia) 1 were cultured in DMEM medium supplemented with 10 % fetal calf serum, 10 mm HEPES and 100 U/ml penicillin/streptomycin at 37 C under 5% CO 2. Growth medium and supplements for cell culture were purchased from Gibco-Invitrogen (Carlsbad, USA). Cells were transfected in 6-well plates at 60% of confluence by means of the Oligofectamine transfection reagent (Invitrogen), following the manufacturer s instructions. The following sirnas were employed to downregulate the mitochondrial phosphate carrier (PiC) and mitofusin 2 (Mfn-2): PiC1, sense 5 - CUGGCGCACAUCACUAUAUdTdT-3 ; PiC2, sense 5 - CCAGGUUAUGCCAACACUUdTdT-3 ; Mfn-2, sense 5 - CGGCAAGACCGACUGAAAUdTdT-3. As a control, an irrelevant sirna was employed (sense 5'-GCCGUAUGCCGGUUAAGUdTdT-3'). 2 To induce cell death, cells were treated with 500 nm staurosporine (Sigma-Aldrich, St. Louis, USA) or with 500 ng/ml α-cd95 monoclonal antibody clone 7C11 (Immunotech, Prague, Czech Republic) plus 100 µg/ml cycloheximide (Sigma-Aldrich), for 6 hours. Immunofluorescence microscopy and cytofluorometry. Immunofluorescence experiments were performed as previously described. 3 Mitochondria were visualized by staining live cells with MitoTracker Orange (Molecular Probes- Invitrogen), following the manufacturer s recommendations, or by using a monoclonal antibody that specifically recognize the respiratory chain complex III core 2 subunit (A11143, Molecular Probes-Invitrogen) revealed with Alexa Fluor 568-coupled secondary antibodies

2 (Molecular Probes-Invitrogen). Acquisition was performed on a Leica DMIR2 inverted fluorescence microscope (camera Leica DFC300FX, acquisition software Leica FW4000). To measure mitochondrial transmembrane potential and cell viability, cells were incubated for 20 min at 37 C with 40 nm 3,3'-dihexyloxacarbocyanine iodide (DiOC 6 (3), from Molecular Probes-Invitrogen) plus 1 µg/ml propidium iodide (PI, from Sigma-Aldrich), immediately followed by cytofluorometric analysis on a FACScan cytofluorometer (Becton Dickinson, San Jose, USA). 4 RT-PCR. Total RNA was isolated by means of the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer s instructions, followed by generation of total cdna with the ImProm-II TM Reverse Transcription System (Promega, Madison, USA). Primer pairs specific for the isoform B of PiC (gene name: slc25a3; accession number NM_ ; forward primer 5 -GTTCTCGTCCGTGGCGCACCTGGCG-3, reverse primer 5 - GGGAACCACAGCAGTGTGTGTCAGACCACAA-3 ) and β-actin (as control, primers G5740 purchased from Promega) were used for semi-quantitative PCR amplification (25 cycles, linear range, PCR Master Mix from Promega). Finally PCR products were separated on 2% agarose gels containing 0.1 µg/ml ethidium bromide (from Sigma-Aldrich) and visualized on a standard UV transilluminator. SUPPLEMENTAL REFERENCES 1. Goldmacher VS et al. A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2. Proc Natl Acad Sci USA 1999; 96: de La Motte Rouge T et al. A novel epidermal growth factor receptor inhibitor promotes

3 apoptosis in non-small cell lung cancer cells resistant to erlotinib. Cancer Res 2007; 67: Castedo M et al. Quantitation of mitochondrial alterations associated with apoptosis. J Immunol Methods 2002; 265: Criollo A et al. Mitochondrial control of cell death induced by hyperosmotic stress. Apoptosis 2007; 12:3-18

4 A sirna Mfn-2 + UNR sirna Mfn-2 + PiC B p < p < % of cells with fragment ed mitochondria sirna Mfn-2 sirna PiC Suppl. Fig. 1

5 LEGENDS TO SUPPLEMENTAL FIGURES Supplemental Figure 1. A-B. sirna-mediated depletion of PiC prevents mitochondrial fragmentation induced by the knockdown of mitofusin 2 (Mfn-2). A549 cells were transfected with a control, irrelevant sirna (UNR) or with sirnas for the downregulation of Mfn-2 and PiC in the indicated combinations for 48 hours, then labelled with MitoTracker Orange for the evaluation of mitochondrial morphology. Panel A reports representative pictures of cells exhibiting normal () or near-to-normal (sirna Mfn-2 + PiC) mitochondria, in contrast with cells displaying mitochondrial macrofragmentation (sirna Mfn-2 + UNR). Scale bars represent 2 µm. Panel B depicts the percentage of cells with fragmented mitochondria, as assessed in a population of at least 300 cells (mean ± STE; n = 3). Paired Student s t tests were performed to evaluate statistical significance.