Supplemental Material s26
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Supplemental Material
Cell lines and Reagents. The pcDNA, TrkAI and TrkAIII stable transfected SH-SY5Y NB cell lines have been described previously (Tacconelli et al., 2004) and were grown in DMEM supplemented with
10% fetal calf serum and appropriate antibiotics (Zeocin, penicillin and streptomycin). Cell culture reagents, MG-132, geldanamycin, 17-(allylamino)-17-demethoxygeldamanycin (17-AAG), 17-
(dimethylamino ethylamino)-17-demethoxy geldanamycin (17-DMAG), cytochalasin D, nocodozol, proteinase inhibitor Cocktail, Bis-benzimide tri-hydrochloride, propidium iodide, HistodenzTM and NGF were from Sigma-Aldrich (St. Louis MO). The pan Trk inhibitor CEP-701 was from Cephalon Inc.
(West Chester, PA). VectorMountTM mounting medium was from Vector Laboratories (Berlingame,
Ca).
Antibodies. Polyclonal anti-TrkA (C14) and monoclonal anti-phospho-tyrosine (pY99), Bcl-2, calnexin, ATF-6 and Hsp90/ F- antibodies were from SantaCruz (Santa Cruz, Ca); polyclonal anti-calreticulin, grp78/BiP, Grp-94 and -tubulin antibodies were from Sigma (St. Louis, MO).
Polyclonal anti-COP I, COP II and TGN46 antibodies were from Abcam (Cambridge, UK). The polyclonal anti-Hsp90 antibody (Ab-1) was from Thermo Scientific (Runcorn, UK). The monoclonal anti GM130 antibody was from BD Bioscience (San Jose, Ca). FITC and Texas red-conjugated secondary anti mouse and anti-rabbit IgG antibodies were from Jackson Immune Research (Bar
Harbor, Maine).
TrkAIII Peptide Nucleic Acid (PNA) synthesis. PNA monomers were from (PRIMM Milan, IT); amino-acids, activators and resins were from Novabiochem (Nottingham, UK); acetonitrile (ACN) was from Reidel-deHaën (Sleeze, GE), dry N, N-dimethylformamide (DMF) was from Lab-Scan
(Stillorgan, Irl). All other reagents were from Fluka (St. Lois, MO). Solid- phase PNA synthesis was performed on an ExpediteTM 8909 Nucleic Acid Synthesis System ABI. LC-MS analyses were performed on a LC-MS Thermo Finnigan with an electrospray source (MSQ) on a Phenomenex Jupiter
1 5μ C18 300Å, (150x460) column. A Phenomenex Jupiter 10 Proteo 90Å (250x10mm 10micro) column was used for purification on a semi-preparative scale. Peptides were synthesized on a PAL-
PEG resin (0.16 mol/g). Briefly, amino acids (10 eq.) were pre-activated in HOBT/HBTU (9.8 eq)
0.5M in DMF and DIPEA (30 eq) and coupled for 15 minutes. The resin was washed with DMF (2 flow wash 25s) and capping performed in acetic anhydride/DIPEA/DMF (15/15/70 v/v/v) for 5 minutes, followed by two 25s flow washes with DMF. Peptides were de-protected in piperidine/DMF
(20/80 v/v) for 5 minutes, resin washed with DMF (2 flow wash 25s), transferred into a small reaction vessel for PNA oligomer assembly. PNAs were synthesized on a 2mol scale in an Expedite 8909 synthesizer using standard protocols. Double coupling was carried out on poly-purine tracts (double coupled bases are underlined in the sequences). At the end of the synthesis resins were washed with
DCM and vacuum dried. PNA and the peptide-PNA conjugates were cleaved from the resin and de- protected in a solution of TFA/TIS/m-cresol (78/2/20) for 90 minutes at room temperature to obtain
KKAA-TrkAIII (KKAA)4-GGCCGGGACACA, which was purified by HPLC in a 5 minute gradient of 7% isochratic water (0.1% TFA), followed by a 30 minute gradient of acetonitrile (0.1% TFA) in water (0.1% TFA) from 7 to 35%, eluted at 22.2 minutes, identified by electro-spray mass analysis and lyophilised three times to remove excess TFA. For cell culture, lyophilised TrkAIII-PNA was solubilised in sterile water, diluted in complete culture medium to 10M and added to cell cultures.
Knockdown of TrkAIII expression was verified by RT-PCR and Western blotting.
HistodenseTM Ultracentrifugation Fractionation of Internal Cell Membranes. Briefly, cells were incubated with cytochalasin D (1g/ml) and nocodozol (0.1M) for 1 hour at 37°C prior to harvest, scraped into cold PBS, washed in homogenisation buffer (130mM KCl, 5mM MgCl2, 25mM Tris-HCl pH 7.3) and homogenised in 500l homogenisation buffer containing proteinase inhibitor cocktail
(1mM 4-(-2 aminoethyl) benzenesulfonylfluoride hydrochloride, 20M leupeptin, 15M pepstatin and
15M chymostatin). Homogenates were centrifuged for 5 minutes at 1000xg at 4°C, supernatants
2 collected and re-centrifuged at 1000xg for 5 minutes at 4°C, to give the post nuclear supernatant (PNS).
The PNS was adjusted to 500l in homogenisation buffer and layered on top of a step-gradient from bottom to top, of 500l of 40%, 1ml of 20%; 1ml of 17.5% and 1ml of 15% HistodenzTM (Sigma-
Aldrich) in homogenisation buffer. Gradients were ultracentrifuged at 4°C in a Sw55Ti rotor for 60 minutes at 100,000xg and 500l fractions collected (top to bottom) and mixed with 125l of 5x detergent buffer (5%NP-40, 2.5% deoxycholate, 0.5% SDS, 250mM Tris-HCl pH 8.0). Fractions were either analysed directly by Western blot or subjected to immunoprecipitation/Western blot.
Immunoprecipitates were washed with RIPA buffer (1% NP-40, 0.5% deoxycholate, 0.1% SDS,
150mM NaCl and 50mM Tris-HCl pH 8.0); twice with high salt buffer (0.2% SDS, 1% TritonX-100,
5mM EDTA, 500mM NaCl and 50mM Tris-HCl pH 8.0) and once with TEN (50mM Tris-HCl pH 8.0;
5mM EDTA and 150mM NaCl), prior to reducing SDS PAGE/Western blotting.
RT-PCR and primers. TrkA I/II and TrkAIII, forward primer 5’-
AACCTCACCATCGTGAAGAGTGGT-3’ and reverse primer 5’-GGTTGAACTCGAAA
GGGTTGTCCA-3’; Hsp90forward primer 5’-GAGCAGTACGCTTGGGAGTC-3’ and reverse primer 5’-TATCGTCGGGATTTCTGGTC-3’; Hsp90forward primer 5′-GTCTGGGTATC
GGAAAGCAAG-3’ and reverse primer 5′-CTGAGGGTTGGGGATGATGTC-3’; Grp94, forward primer 5′-GCTCGACTCGAATTCCAAAG-3′ and reverse primer 5′-TTTGTCAGGGGTCTT
TCACC-3; NF-M, forward primer 5’-GAGCGCAAAGACTACCTGAAGA-3’ and reverse primer 5’-
CAGCGATTTCTATATCCAGAGCC-3’; Neuro-D, forward primer 5’-ATCCCAACCCACCACCAA
CC-3’ and reverse primer 5′-CAG CGG TGC CTG AGA AGA TT-3’; XBP-1 forward primer 5’-
GAAACTGAAAAACAGAGTAGCAGC-3’ and reverse primer 5’-GCTTCCAGCTTGGCTGATG
-3’ and GAPDH: Forward primer 5'-CGGAGTCAACGGATTTGGTCGTAT-3' and reverse primer 5'-
AGCCTTCTCCATGGTGGTGAAGAC-3', used to control RNA quality and quantity. RT-PCR products were resolved by 1.5% agarose gel electrophoresis.
3 Immunoprecipitation and Western Blots. Cells were extracted in lysis buffer (PBS containing 0.5% sodium deoxycholate, 1% NP40, 0.1% SDS, 1mM sodium orthovanadate, 1mM PMSF, 1g/ml of pepstatin A and Aprotinin), protein concentrations calculated by Bradford Assay (Sigma-Aldrich) and extracts pre-cleared with 1g pre-immune IgG (1 hour at 4°C) and 20l protein A Sepharose (Fast
Flow, Sigma) for 20 minutes at 4°C. For immunoprecipitation, pre-cleared extract (for TrkA immunoprecipitation 200-500g for stable transfectants and 3-5mg for non-transfectants) were incubated with primary antibody at a concentration of 0.1-1.0g/500g total protein for 2-16 hours at
4°C. Following incubation, 20l Protein A Sepharose (Fast flow, Sigma-Aldrich) in lysis buffer, was added and reactions incubated for 30 minutes at 4°C. Protein sepharose/IgG conjugates were collected by centrifugation (10,000xg for 5 minutes), washed 3 times in lysis buffer, resuspended in SDS-PAGE sample buffer and subjected to reducing SDS-PAGE/Western blotting. Proteins were transblotted by electrophoresis onto Hybond C+ nitrocellulose membranes (Amersham Int. UK) and membranes air- dried. Non-specific protein binding site were blocked by 2 hours incubation in 5% non-fat milk in
TBS, prior to incubation for 2-16 hours with primary antibodies diluted in blocking solution at 4°C.
Following incubation, membranes were washed in TBS, incubated for 20 minutes with secondary
HRP-conjugated antibody diluted in blocking solution and immunoreactive species detected by chemiluminescence reaction, as directed (Amersham Int., Bedford, UK).
Metabolic Labelling of TrkAI and TrkAIII. Briefly, 3-4 million cells per time point, pre-incubated for
8 hours in the presence or absence of 1M GA, were incubated for 2 hour in methionine and cystein- free medium prior to exposure for 30 minutes to 100Ci/ml S35-labelled methionine/cystein (Perkin-
Elmer). Cells were washed with pre-warmed complete medium and chased in excess (1mM) cold methionine/cystein (Sigma-Aldrich), for different times. Aliquots at each time point were incubated for 45 minutes on ice with ice cold PBS containing 0.5mg/ml Sulfo-NHS biotin (Pierce), washed 4 times in ice cold PBS containing lysine to remove excess unbound biotin, and proteins extracted in ice
4 cold lysis buffer (20mM Tris-HCl, pH8.0; 137mM NaCl; 2mM EDTA; 10% glycerol; 1% NP-40;
20M leupeptin; 1mM sodium vanadate; 1mM Pefabloc; 0.15U aprotinin; 1mM -glycerophosphatase and 6mM sodium fluoride). Extracts were clarified by centrifugation at 12,000xg for 10 minutes and immunoprecipitated using anti-TrkA antibody (C14). Immunoprecipiates collected using protein sepharose A beads (Fast Flow, Sigma), were washed twice in lysis buffer and eluted by boiling for 10 minutes in 10% SDS. Eluted immunoprecipitates were divided, one half processed for Western blotting and the remainder diluted 100 fold in SDS-free RIPA buffer to dilute SDS to 0.1%, prior to being subjected to a second precipitation using 50l of Streptavidin beads (Pierce), to obtain the cell surface biotinylated TrkA fraction. Washed cell surface Streptavidin-Biotin precipitates and total cell immunoprecipitates were subjected to regular reducing SDS-PAGE and dried gels subjected to autoradiography.
TrkAIII Adenoviral and the TrkAIII Y670/674/675F mutant expression vectors. Briefly, pAd/CMV/TrkAIII-V5-DEST adenovirus, amplified in 293A cells (Invitrogen), was transduced into
SH-SY5Y cells at an appropriate multiplicity of infection and TrkAIII protein expression confirmed by
Western blotting. The TrkAIII cDNA bearing phenylalanine (F) substitutions of tk loop tyrosines (Y)
670, 674 and 675 was constructed from pcDNA-TrkAIII (Tacconelli et al., 2004) by substituting a
370bp EcoRV digestion fragment with a PCR product generated using the Y670F, Y674F/Y675F mutant forward primer 5’-AGCAGGGATATCTtCAGCACCGACTttTtC CGTGTG-3’ and the reverse primer 5’-GCCACTGTGCTGGATATCTGCAGAATTC-3’. Y670, 674 and 675 mutations were confirmed by sequencing in an ABI prism automated DNA sequencer. Mutated TrkAIII cDNA was cloned into the pcDNA3.1 mammalian expression vector and SH-SY5Y cells were stably transfected with the pcDNA3.1-Y670/674/675F mutated TrkAIII (muTrkAIII) using lipofectamine, as directed by the manufacturer (Invitrogen) and stable transfectants isolated by zeocin resistance and characterised, as previously described (Tacconelli et al., 2004).
5 Luciferase reporter gene Assays. Briefly, stable transfected SH-SY5Y cells grown to 50% confluence in 10cm plates were transfected overnight with either 2g of minimal cfos-promoted enhancer-less
PoFLuc-GL3 or 5xATFPoFluc-GL3 reporter gene (Wang et al., 2000), together with 1g of pcDNALacZ expression vector using Fugene, as described by the manufacturer (Roche, IN).
Following 24 hour transfection and subsequent 12 hour treatment with 1M GA, cells were lysed in
RIPA buffer, assayed for -galactosidase activity, as previously described (Eustice et al., 1991), normalised for -galactosidase activity and assayed for luciferase activity using a firefly luciferase kit, as directed by the manufacturer (Promega). Results are expressed as the mean (+s.d.) luciferase activity from quadruplet determinations, in four independent experiments.
MTS Proliferation and Survival Assays. For proliferation assays SH-SY5Y transfectants were plated onto 96-well mictrotitre plates, at a concentration of 1,000 cells per well (6 wells per experimental point), allowed to attach for 6 hours, washed then incubated with either complete medium alone or with different concentrations of GA, for 24, 48 and 72 hours at 37°C. Following incubation 20l of MTS reagent (Promega) was added to each culture, incubated for 3 hours at 37°C and absorbance read at
492nm in a microtitre plate-reader (Biotek Instr.). For survival assays, SH-SY5Y transfectants were plated at a concentration of 10,000 cells per well onto 96 well microtitre plates (6 wells per experimental time point), allowed to attach for 6 hours, washed then incubated with complete medium alone or with different concentrations of GA for 24 hours. MTS reagent addition and measurement of absorbance at 492nm, was as described above. siRNA Hsp90 Knockdown. Briefly, TrkAI and TrkAIII transfectants seeded onto 24 well culture plates (Costar), at 5,000 cells/ml in complete medium were allowed to attach for 6 hours. Cells were washed 3 times in serum-free medium and duplicate cultures incubated for 6 days in serum-free Accell
SMART Pool transfection medium alone or containing 1M of Hsp90Hsp90 or Grp94 siRNAs.
Medium was replaced on day 3 and knockdown was assessed by RT-PCR and Western blotting.
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