Supplemental Figure S1 Tonne et al.

Transcription

1 Supplemental Figure S1 Tonne et al. A 1 26 Signal peptide GFP 1 76 NT-proBNP 1 GFP NT-proBNP GFP BNP32 SP-GFP NTpro-GFP probnp-gfp B SP-GFP NTpro-GFP probnp-gfp IB: α-gfp (kda) Fig. S1. Supranuclear localization of GFP-tagged BNP. (A) Schematic representation of C-terminally GFP-fused BNP constructs. (B) Verification of the expression of the GFP-fused constructs in HL-1 cells by an anti-gfp MAb. (C) 293T and HL-1 cells were transfected with the expression plasmids. Forty eight hours after transfection, cells were fixed and the GFP signals were analyzed by confocal microscopy. Nuclei were counter-stained by DAPI (blue). D. Co-localization of the supranuclear GFP-tagged BNP signals with the Golgi apparatus marker Giantin. Forty eight hours after transfection, cells were fixed and the Golgi apparatus was detected by anti-giantin antibody. Localization of the GFP signals (green) and the Golgi marker signals (red) were analyzed by confocal microscope. E. No apparent co-localization of GFP-tagged BNP (green) with early endosome marker EEA1 or mitochondria marker AIF (red). C D NTpro-GFP GFP 293T HL-1 GFP DAPI GFP SP-GFP NTpro-GFP probnp-gfp Giantin DAPI Merged E NTpro-GFP EEA1 probnp-gfp AIF Accumulation of GFP-tagged B-type natriuretic peptide proteins in Golgi apparatus. The intracellular trafficking of B-type natriuretic peptide was studied using green fluorescent protein (GFP)-tagged B-type natriuretic peptide constructs (Supplementary Fig. S1A). After verification of their expression by immunoblotting (Supplementary Fig. S1B), we tested their subcellular localization in human kidney 293T and murine cardiomyocyte HL-1 cells. Expression of a control, GFP-only construct resulted in diffuse cytoplasmic signals. Although expression of SP-GFP, a GFP-fused with the 26 amino acid signal peptide sequence, was toxic in 293T cells, supranuclear green fluorescent signals were observed in the SP-GFP-expressing HL1 cells, suggesting that the signal peptide controls the supranuclear accumulation of intracellular B-type natriuretic peptide. NTpro-GFP and probnp- GFP showed a clustered supranuclear localization of green fluorescent signals with small discrete cytoplasmic body signals in 293T and HL-1 cells (Supplementary Fig. S1C). To determine the region where BNP-GFP proteins accumulate, we stained the BNP- GFP-expressing cells with subcellular localization markers. The supranuclear BNP-GFP signals co-localized frequently with Golgi marker giantin signals (Supplementary Fig. S1D). No typical colocalization was observed between the BNP-GFP fusion proteins and the endosomal (EEA1) or the mitochondrial (AIF) markers (Supplementary Fig. S1E), indicating that intracellular B-type natriuretic peptide trafficking is directed through the Golgi apparatus.

2 Supplemental Figure S2. Tonne et al. IP: α-bnp1-32 polyclonal (Phoenix) (Abcam) BNP1-32 Intracellular Secreted Intracellular Secreted HMWproBNP probnp BNP1-32 IB: mab 24C5 Fig. S2 Selective secretion of glycosylated probnp in 293T cells. PreproBNP-expressing constructs were transfected in 293T cells, and cell lysates and filtered culture supernatants were analyzed for the molecular forms of BNPs using anti-bnp1-32 rabbit polyclonal antibodies from Phoenix and Abcam. Anti- BNP1-32 monoclonal antibodies 24C5 was used to detect immuno-reactive BNP in pulled-down cell lysates and supernatants. Selective secretion of glycosylated probnp was confirmed by IP with two anti-bnp32 polyclonal antibodies.

3 SUPPLEMENTAL MATERIAL EXPANDED MATERIALS Cell culture and plasmids. HEK 293T cells were maintained in Dulbecco s modified Eagle s medium supplemented with 10% calf serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. A murine atrial cardiomyocyte cell line, HL-1, was kindly provided by Dr. William C. Claycomb (Louisiana State University Medical Center, New Orleans) and cultured in Claycomb s medium with 10% FBS, 100 µm norepinephrine, and 4 mm L-glutamine on 0.02% gelatin/fibronectin-coated flasks or plates. Normal human cardiomyocytes (48-year-old female, Caucasian, ventricle-derived) were purchased from Promocell (Heidelberg, Germany), and maintained under manufacture s guidelines using the Myocyte Growth Medium and Cell Detach Kit supplied upon purchase. All cells were kept at 37 C with 5% CO2. The codon-optimized human was synthesized by GenScript, and sub-cloned into the BamHI-NotI sites of a lentiviral vector psin-csgwdlnoti 1 and psin-gfp-ubem 2, resulting in psin-bnp and psin-bnp-ubem, respectively. The -expressing plasmid paav- was generated by sub-cloning the BamHI-XhoI fragment of the psin-bnp into a mammalian expression plasmid, paav-mcs (Stratagene). paav-based NT-proBNP-deleted mutant, paav-sp-bnp, was generated by extension PCR. The GSS-proBNP construct, which has the gaussia luciferase secretory signal in the place of BNP signal peptide, was generated by introducing the secretory signal sequence by PCR and re-cloned into the paav-. T71A mutant was generated by introducing the mutation by site-directed mutagenesis (Quikchange, Stratagene). The C-terminally EGFP-fused BNP constructs were generated by cloning PCR-amplified corresponding BNP sequences in pegfp-n1 (Clontech) in in-frame. Transfection. 293T cells were seeded at 1 x 10 6 cells/well in a 6 well plate, and transfected with FuGene-6 transfection reagent (Roche Diagnostics) based on manufacturer s protocol. Media was changed 8-12 hours post-transfection with

4 fresh DMEM containing 10% FBS and antibiotics. Cells were allowed to grow for 96-hours post-transfection prior to harvesting media and cells. Culture supernatants were collected and passed through a Whatman 0.45 µm filter to remove any cell debris and stored at -80 C until use. Transfected cell monolayers were first rinsed with PBS then lysed in RIPA buffer (50 mm Tris-HCl at ph 7.4, 1% NP-40, 0.25% sodium-deoxycholate, 150 mm NaCl, 1 mm EDTA, 10 µl/ml protease inhibitor cocktail) on ice for 10 min. Remaining cell debris was removed by centrifugation at 4 C for 10 min, and supernatants were stored at - 80 C as cell lysates. FuGene-6 transfection reagents typically resulted in approximately 80% and 10% transfection efficiency in 293T and HL-1 cells, respectively. Lentiviral Vector Production HIV vectors were produced by transient transfection of 293T cells using FuGene6 (Roche, Indianapolis, IN, USA) with a weight ratio of 2:1:1 of vector (psin-bnp or psin-bnp-ubem) to packaging (pex-qv) to VSV-G plasmids. Six hours after transfection, the medium was changed to complete Dulbecco s modified Eagle s medium. The viruses were harvest 48 and 72 h after transfection. The harvested viruses were filtered through 0.45 µm filter membrane (Millipore) and concentrated by ultra centrifugation as described previously 3. Lentiviral vector transduction of HL-1 and primary human cardiomyocytes has been repeated at least three times and typically resulted in over 90% transduction efficiency. Immunoblotting. Culture supernatants and cell lysates were mixed 1:1 with Laemmli buffer containing 2-ME. The samples were boiled at 99º C for 5-10 min, kept on ice for few min, and then loaded into a 15% SDS-polyacrylamide gel and then transferred to PVDF membrane. Immuno-reactive BNPs were detected using a HRP conjugated mouse monoclonal 24C5 antibody to the N-terminal amino acids of human BNP1-32 (1:500, Abcam) and mouse monoclonal 50E1 to amino acids of human probnp1-108 (1:500, Abcam). Additional antibodies we used in our experiments include: rabbit polyclonal anti-bnp1-32

5 (Phoenix and Abcam), rabbit polyclonal anti-probnp (Phoenix), rabbit polyclonal to corin spacer region (1:5000, Abcam) and mouse monoclonal β-actin (1:1000, Sigma). Immunostaining. For BNP localization we utilized HEK 293T, HL-1 mouse cardiomyocytes, and primary human cardiomyocytes. The cells were seeded on 8-well chamber slides (Nunc) treated with 0.02% gelatin/fibronectin and allowed to grow for 48-hours post-transfection before washing with PBS and fixing in 4% paraformaldehyde for 20 min at room temperature. The cells were then permeabilized with PBS containing 0.3% Triton-X for 10 min, washed and then blocked for 1-hour in PBS containing 5% FBS at room temperature. Primary antibody treatment was done overnight (~14 hr) in humidity chambers to prevent drying. Cells were washed three times with PBS and then be incubated with Alexa Fluor 488 goat anti-rabbit or mouse IgG (H+L) (Invitrogen) for 60 min in the presence of DAPI. Cells were then observed with a Zeiss LSM 510 confocal laser scanning microscope and the images were analyzed with Zeiss imaging software. Antibodies and concentrations used for immunocytochemistry include: 50E1 mouse monoclonal to human BNP-32 (1:200, Abcam), mouse monoclonal 9B6 to giantin of the Golgi bodies (1:250, Abcam), EEA1 rabbit polyclonal to early endosomes (1:200, Cell Signaling Technology), AIF rabbit polyclonal against Apoptosis Inducing Factor (1:200, Cell Signalling Technology), mouse monoclonal β-actin (1:200, Sigma), and mouse monoclonal to α-actinin (1:200, Sigma). Immunoprecipitation. Culture supernatants were collected and prepared as described above. Cells were grown in 6-well format and lysed in 500 µl of RIPA buffer. For immunoprecipitation, 500 µl and 1 ml were used for lysates and culture supernatants, respectively. Each sample was initially incubated with µl of agarose beads (protein A or protein G) at 4 ºC rotating for 60 min. The beads were then centrifuged down, removed and antibody against the epitope of interest was added. Samples were rotated over-night at 4 ºC, before adding 30

6 µl agarose beads to collect the antibody-antigen complex. The beads were gently centrifuged down and the remaining supernatants were removed. The bead pellets were gently re-suspended in 500 µl of RIPA buffer, gently centrifuged down and wash buffer was removed. This was repeated for a total of 5 washes. After the final wash the beads were once again centrifuged down, the wash buffer removed, and Laemmli sample buffer containing 2-ME was added 1:1. The samples were boiled at 99 ºC for 5-10 min before running on SDS- PAGE. Tandem-MS analysis. Pre-proBNP-expressing 293T supernatants were enriched through immunoprecipitation with anti-probnp monoclonal antibody 15F11 as described above. Eluted proteins were then separated in a SDS-PAGE gel, and silver-stained for protein visualization. Proteins with 18 to 22 kda and the corresponding areas in the control 293T supernatant lane were excised and analyzed using tandem MS. Peptide assignments from the tandem MS spectra were accepted if they could be established at greater than 99.9% probability as specified by the PeptideProphet algorithm. Keratin was excluded as it is known as a common contaminant in proteomic analysis. The identified BNP-derived peptides were listed in Fig. 4C. No BNP-derived peptides were identified in the control. 1. Nelson TJ, Martinez-Fernandez A, Yamada S, Mael AA, Terzic A, Ikeda Y. Induced pluripotent reprogramming from promiscuous human stemness related factors. Clin Transl Sci. 2009;in press. 2. Hasegawa K, Nakamura T, Harvey M, Ikeda Y, Oberg A, Figini M, Canevari S, Hartmann LC, Peng KW. The use of a tropism-modified measles virus in folate receptor-targeted virotherapy of ovarian cancer. Clin Cancer Res. 2006;12(20 Pt 1): Noser JA, Towers GJ, Sakuma R, Dumont JM, Collins MK, Ikeda Y. Cyclosporine increases human immunodeficiency virus type 1 vector transduction of primary mouse cells. J Virol. 2006;80(15):