Supplementary Methods

Transcription

1 Supplementary Methods Antibodies Rabbit primary antibodies used in this study included anti-phospho-p44/42 MAPK (Thr202/Tyr204), anti-phospho-mek1/2 (Ser217/221), anti-phospho-c-raf (Ser259), antiphospho-sapk/jnk (Thr183/Tyr185), anti-phospho-p38mapk (Thr180/Tyr182), antiphospho-akt (Ser473), (Cell Signaling Technology), anti-human ErbB2 antibody (Santa Cruz), anti-human ErbB3 (Santa Cruz), anti-erbb1/egfr, anti-phospho-erbb2 (Tyr1248) (Cell Signaling Technology), and anti-p0/mpz (a gift from Marie Filbin, Hunter College, New York). Mouse monoclonal antibody used were anti-n-terminal extracellular domain of human ErbB2 (NeoMarkers, Lab Vision Corporation), anti-phospho-tyrosine (Cell Signaling Technology), anti-his-tag antibody (Qiagen), anti-pgl-1 (a gift from Dr. Arend Kolk, Royal Tropical Institute, Amsterdam, The Netherlands), anti-brdu (Roche Molecular Biochemicals), anti-ß-actin (Sigma), anti-laminins 2, 4. The ErbB2 inhibitory antibodies 4D5 and 2C4 were obtained from Genentech Inc. CA. Western blot analysis and immunoprecipitations Total protein extracts were prepared by harvesting the cells in cell lysis buffer containing 62mM Tris-HCl ph 6.8, 2% SDS, 10% Glycerol, 50mM DTT and 0.2% protease inhibitors cocktail (Sigma). Lysates were cleared by centrifugation and the protein content was determined according to the BCA protocol (PIERCE Chemical, Rockford IL). 20µg of total cell extracts were separated by SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell). The bound antibodies were detected using species-specific secondary antibodies coupled to HRP (Cell Signaling Technology) followed by enhanced chemiluminescence development (PIERCE Chemical, Rockford IL). Protein expression intensity was normalized to ß-Actin and quantified with a densitometer (Alpha Innotech Corporation) using the Alpha Imager 2200 v.5.5 software. Membrane fractions were prepared using standard protocol as described 1. Briefly, cells were incubated in a hypotonic buffer (10mM Tris-HCl ph 8.0, 1mM EDTA, 1mM PMSF, 1mM phenanthroline, 1mM iodoacetamide) for 10min at 4 0 C, and then scrapped and the membranes were pelleted by centrifugation at 40,000 rpm for 20min at 4 0 C and directly resuspended in 1 X Lammeli buffer. For immunoprecipitations, cells 1

2 were lysed in a buffer containing 250 mm NaCl, 0.1% NP-40, 50 mm HEPES ph 7.0, 5 mm EDTA and 0.2% protease inhibitors cocktail (Sigma). 100µg of protein extracts were incubated overnight (o/n) at 4 0 C with 50µl of ErbB2 polyclonal antibody (Santa Cruz) followed by incubation with protein A-agarose beads (PIERCE) for 2h at 4 0 C. Beads were washed extensively in immunoprecipitation buffer, resuspended in 50µl of sample buffer and then subjected to SDS-PAGE. Bound antibodies were detected using speciesspecific secondary antibodies coupled to HRP (Cell Signaling Technology) followed by enhanced chemiluminescence development (PIERCE). Kinase inhibition assays For the inhibition of Erk1/2, myelinated Schwann cell-neuron co-cultures and Schwann cell mono-cultures were first preincubated with µm of MEK1/2 inhibitor UO126 (Cell Signaling Technologies) for 30 min and then M. leprae suspension in media containing U0126 were added for 15 min. Control cells were incubated for the same time period with the vector DMSO. For the inhibition of ErbB2 activation, Schwann cell-neuron cocultures were incubated with 1mg/ml PKI166 (Novartis) for 1h prior to M. leprae treatment and then for 15 min with M. leprae (The concentration of PKI166 used did not cause apoptosis or cell toxicity). As a control, myelinated cultures were treated with 0.01% DMSO. For the inhibition of M. leprae-induced Erk1/2 in human primary Schwann cells we used Hereceptin/4D5 antibody as described below. To determine the effect of laminin-2 on M. leprae-induced phosphorylation of ErbB2 and demyelination, 1X10 8 /ml M. leprae were incubated with 100µg/ml Laminin-2/4 for 1 hour at room temperature as described previously 2. Treated M. leprae were added to myelinated Schwann cell neuron co-cultures for 30 minutes and then removed and the cultures were washed three times with PBS. Total protein extracts, ErbB2 immunoprecipitations and western blotting were performed as described below. Demyelination was evaluated as described previously 3. Inhibition of bacterial attachment to SKBR-3 cells by Herceptin antibody. To inhibit the M. leprae binding to native ErbB2 on the surface of SKBR-3 cancer cells, and human Schwann cells we used 20µg/ml 2C4 or 4D5 (Herceptin) anti-erbb2 inhibitory antibodies (obtained from Genentech Inc. CA). The cells were pre-incubated with the 2

3 antibodies for 30 min and also included during infection with 1X10 8 /ml M. leprae. HeLa cells were used as a control. To determine the inhibition of M. leprae attachment to SKBR-3 cells, cells were stained with anti-pgl-1 MAb that specifically detect M. leprae 4 and visualized with a Zeiss LSM 510 confocal microscope. Control cells were trated with 20µg/ml mouse IgG1. Cell bound bacteria was quantified by analyzing 10 different fields from three independent experiments and the results are presented as average percentages ± standard deviation. PCR amplification of ErbB2 extracellular domain and plasmid construction Total RNA from human Schwann cells (5µg) was used as a template for reverse transcription using Superscript II reverse transcriptase (Invitrogen) and a combination of oligo-dt and random hexamers. Resulting cdna (2µg) were used to amplify the region coding for the extracellular domain of ErbB2 (amino acids: 1-652) in a PCR over 35 cycles of 30 seconds at 94 0 C, 30 seconds at 61 0 C and 2 minutes at 72 0 C. The primers used (sense: 5 -ACCTCTTCGATGGAGCTGGCGGCCTTG-3, anti-sense: 5 - ACCTCTTCGAAGCGTCAGAGGGCTGGCTCTCT-3 ) contained the Eam1104 I recognition sequence (underlined) for directional cloning into the pdual-gc expression vector (Stratagene) that allows high level His-tagged protein expression in mammalian systems. The resulting construct (pdual ErbB2Ex ) was confirmed to contain the region coding for the extracellular domain of ErbB2 in frame, by restriction mapping and sequencing. Transfections and purification of the recombinant extracellular domain of ErbB2 (rerbb2ex) COS-7 cells were transiently transfected with the pdual ErbB2Ex construct using Lipofectamine-2000 (Invitrogen) reagent. After 48h of transfection the supernatants were collected, centrifuged to remove any cellular debris and coupled to TALON affinity resin (BD Biosciences) according to the manufacturer s instructions. The purified protein was eluted with 100mM Imidazole and dialyzed overnight in PBS. The expression, purity and activity of rerbb2ex were analyzed by SDS-PAGE and immunoblotting using anti-his-tag and anti-extracelluar domain of ErbB2 antibodies as well as binding of rerbbex to Hereceptin/4D5 antibody (see main text and Suppl.Fig. 2). 3

4 Generation of Lenti-ErbB2 EXTR/TRM and transfection of 32D cells The portion of human ErbB2 gene containing the extracellular and transmembrane domains (EXT/TRM) was PCR amplified from primary human Schwann cell RNA using the following primers. Forward: 5 -ATGGAGCTGGCGGCCTT-3 ; Reverse: 5 - GATGAGGATCCCAAAGAC-3. The PCR product was purified and directionally TOPO cloned into the plenti6/v5-d-topo plasmid (Invitrogen) according to manufacturer s instructions. 3µg of plenti6/v5-d-erbb3 was used together with 9µg of ViraPower Packaging Mix (Invitrogen) to transfect 5 X FT cells with Lipofectamine after transfection, the supernatant containing the virus (Lenti-ErbB2 EXTR/TRM ) was collected, centrifuged and stored at 80 0 C. 32D cells were transfected with Lentivirus- ErbB2 EXTR/TRM using a multiplicity of infection (MOI) of 1 for 48h and then processed for immunofluorescence and Western blotting. To assess M. leprae binding to Lenti-ErbB2 EXT/TRM -transfected 32D cells, we prepared cytospins of 1X10 4 cells and fixed them by air-drying overnight at room temperature. After rehydration in PBS, 32D and 32D/ Lenti-ErbB2 EXT/TRM cells were incubated with 1X10 8 /ml M. leprae for 1h and then washed extensively with PBS and processed for immunofluorescence using antibodies to ErbB2 and PGL-1 as described above. Receptor binding assays Specific binding of M. leprae to the recombinant extracellular domain of human ErbB2 was assessed by an ELISA. Briefly, 1.5µg of rerbb2ex was coated overnight onto polystyrene plates (Nunc) in 0.1M Carbonate buffer ph 9.5 at 4 0 C. After blocking in PBS with 5% nonfat dry milk and 0.05% Tween-20, increasing concentrations of M. leprae (5X10 6 /ml 2.5X10 7 /ml) were allowed to bind for 45 min at 37 0 C. Subsequently, plates were extensively washed with PBS and the bound bacteria were detected with anti-pgl- 1 MAb followed by goat anti-mouse-hrp conjugated secondary antibody and Ultra TMB substrate development (PIERCE). The same assay was repeated by using increasing concentrations of ErbB2-Ex (1µg-3µg in triplicates) and pre-evaluated bacterial concentration (3X10 6 /ml). Competitive inhibition studies were performed by incubating increasing concentrations of ErbB2-Ex (1µg-3µg in triplicates) with 3X10 6 /ml M. leprae in the presence or absence of 1µg 4D5 ErbB2 inhibitory antibody (Genentech) for 45 min at 4

5 37 0 C. Each binding assay was repeated four times and the results were analyzed with GraphPad Prizm Software (GraphPad Software) using the nonlinear regression analysis for specific binding and affinity value (Kd) determination. ErbB3 gene silencing by sirna To generate the ErbB3 sirna probe, we searched for ErbB3 specific sequence that has the minimum sequence homologies with other human genes using the BLAST algorithm. The region between nucleotides of ErbB3 showed minimal homology with any known human gene and this specific sequence was selected to generate the double stranded sirna probe. This region was PCR amplified from human Schwann cell cdna, reversed transcribed to RNA, annealed to double-stranded RNA and diced to generate a pool of double-stranded sirnas according to manufacturers instructions (Invitrogen Corporation). The resulting ErbB3 sirnas were transfected into human primary Schwann cells using LipoFectamine 2000 (Invitrogen). For negative control sirnas, we generated LacZ double-stranded sirnas according to the manufacturers instructions (Invitrogen). Specific knockdown of human ErbB3 was verified by Western blotting using antibodies to total ErbB3. Proliferation and apoptotic assays Schwann cell proliferation was measured by 5-Bromo-2 -deoxyuridine (BrdU) uptake using BrdU assay kit (Roche Molecular Biochemicals). BrdU was added to the culture medium during the last hour of the incubation. The percentage of BrdU positive cells as compared to DAPI positive nuclei (BrdU index) was determined by counting 10 contiguous fields and data are presented as average percentages ± standard deviation. Possible induction of apoptosis in Schwann cells after M. leprae treatment was determined using standard TUNNEL assay. Rat and human Schwann cells were treated with M. leprae (1X10 8 /ml) for 12h, 48h and 72h and processed for TUNNEL assay using In Situ Cell Death detection kit (Roche) according to the manufacturers instructions. Data were presented as average percentages ± standard deviation. In addition, expression of other apoptotic markers such as Caspase-3, and poly (ADP-ribose) polymerase (PARP) was analyzed by immunoblotting using lysates of infected cells with antibodies specific to 5

6 Caspase-3, and PARP. Cells treated with Camptothecin for 48h were used as a positive control for Caspase-3, and PARP. References: 1. D.A. Lyons, H. M. Pogoda, M. G. Voas, I. G. Woods, B. Diamond, R. Nix, N. Arana, J. Jacobs, W.S, Talbot, Curr. Biol. 15, 513 (2005). 2. Rambukkana, A., Salzer, J.L., Yurchenco, P.D. & Tuomanen, E.I. Neural targeting of Mycobacterium leprae mediated by the G domain of the laminin-alpha2 chain. Cell 88, (1997). 3. Rambukkana, A., Zanazzi, G., Tapinos, N. & Salzer, J.L. Contact-dependent demyelination by Mycobacterium leprae in the absence of immune cells. Science 296, (2002). 4. Ng, V. et al. Role of the cell wall phenolic glycolipid-1 in the peripheral nerve predilection of Mycobacterium leprae. Cell 103, (2000). 6