2.1 Cell Culture
α–TC1-6 (courtesy of Dr. Bruce Verchere, University of British Colombia, Canada), α–TC3, α–TC-Lenti-IR (courtest of Dr. Michael Wheeler, University of Toronto, Canada), and C2C12 (ATCC) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, 12491-015) while βTC3 cells were maintained in RPMI-1640 medium (Invitrogen, 12633-012), both supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine at 37°C, 95% air, 5% CO2. Cells were passaged weekly and were used at passages 26-45.
Detection of endogenous and over-expressed PANDER by immunoblot and immunohistochemical labeling of PANDER in pancreatic sections was performed using rabbit polyclonal PANDER anti-serum (Alpha Diagnostics, San Antonio, TX). For immuno-fluorescent (IF) labeling, the following antibodies were used: anti-glucagon monoclonal (R&D Systems, MAB1249); anti-glucagon polyclonal (Millipore, 4031-01); anti-FLAG (M2) polyclonal (Cell Signaling Technology, 2368); anti-GM130 monoclonal (BD Transduction Labs, 612008); anti-PDI (Affinity Bioreagents, MA3-019); anti-LAMP-1 (Developmental Studies Hybridoma Bank, DSHB 1D4B), and anti-Chromogranin-A polyclonal (Novus Biologicals, NB110-2475). Secondary antibodies were either Alexa-Fluor dye conjugated [DAR-Alexa 488; DAM-Alexa 594; DAGP-Alexa 647], biotinylated [DAR-biotin, DAM-biotin], or Cy-conjugated [DAM-Cy3, DAR-Cy2]. Tertiary streptavidin-HRP or streptavidin-Alexa-dye conjugates were employed for sequential LSAB-IHC / IF. For assessment of insulin signaling, the following antibodies were employed: pAkt-Thr308 (Cell Signaling, 4056), pAkt-Ser473 (Cell Signaling, 4060), and Akt (Cell Signaling, 9272).
2.3 Transient transfections and nucleofections
Adherent α–TC1-6 cells were transiently transfected with 4 μg PANDER plasmid using Lipofectamine 2000 (Invitrogen, 11668-019) in OPTI-MEM (Invitrogen, 31985-062) according to manufacturer instructions. α–TC1-6 cells in suspension were nucleofected with 2 μg pCMV-PANDER-3-FLAG using the nucleofector kit V (Amaxa Biosystems, VCA-1003) and program G-016 according to manufacturer instructions.
2.4 Cell lysate preparation and western blotting
Adherent cell layers were rinsed with cold Dulbecco’s Phosphate Buffered Saline (DPBS) (Invitrogen, 14040-117) and lysed using Roth’s Lysis Buffer (50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 1 mg/ml aprotinin, and 0.01 mg/ml leupeptin), or 1X Cell Lysis Buffer (Cell Signaling, 9803) supplemented with phosphatase inhibitor cocktail 2 (Sigma, P5726) and 1 mM PMSF. Lysates were stored at −20°C until SDS-PAGE analysis.
For immunoblots, equal quantities of total lysate protein were boiled for 5 minutes in 5X SDS-sample buffer with 15 mg/ml dithiothreitol (DTT) and resolved on 12% polyacrylamide gels. Protein transfer to nitrocellulose or PVDF membranes was carried out and blotted using antibodies listed above and detection performed using ECL Advance Western blotting kit (GE Healthcare, RPN-2135). Immunoblot visualization was perfomed using the FujiFilm LAS-3000 Imaging system. Densitometric analyses were performed using the FujiFilm Multigauge software package.
2.5 Murine islet isolation, dispersion and FACS-sorting
Murine pancreatic islets were isolated from 6-10 week old C57BL/6 mice as previously described (Gao et al., 1999
). Islets were hand-picked, counted, and dispersed using 1.8 U/ml dispase in Hank’s Balanced Salt Solution (HBSS) and gentle trituration. The resulting single-cell suspension was then sorted by fluorescence-activated cell sorting (FACS) as outlined previously (Pipeleers et al., 1985a
2.6 RNA isolation from cell lines and primary islet cell populations
Adherent cell layers were rinsed in cold DPBS, lysed, and RNA isolated using the RNeasy kit (QIAGEN, 74104) according to manufacturer instructions. DNAse treatment of all samples was performed. Equivalent input RNA was used for quantitative Taqman® RT-PCR. For insulin, glucagon, and β–actin amplification, commercially available Gene Expression Assays were used (Applied Biosystems), while for pander, either commercially available or the primer /probes listed below were employed. PANDER forward – 5′-TGCTCGCGGAGCTCATTC 3′, PANDER reverse – 5′-CCAATGCTTCGGATGTTGTAGA-3′, fluorescent (FAM) PANDER probe – 5′-TGACGTGCCCCTGTCCAGCACT-3′.
Primary α, β and non–α/non–β cell enriched populations obtained by FACS sorting of murine islets (as outlined above) were lysed and RNA isolated using the Taqman® PreAmp Cells-to-CT kit (Ambion, 4387299). 500 ng of input RNA was reverse transcribed and pre-amplified for target transcripts. Pre-amplification products were subsequently used in Taqman® qPCR.
2.7 Immuohistochemical labeling of murine islets
Pancreata isolated from male C57BL/6 mice were fixed overnight in 1:10 buffered formalin (Fisher Scientific, SF93-4). Tissue was then processed, paraffin embedded, and 4 μm sections mounted onto ProbeOn™ Plus slides (FisherBiotech, 15-188-52) and slides stored at room temperature until stained. The sections were labeled for PANDER and glucagon using sequential LSAB-DAB and LSAB Immuno-fluorescent staining respectively. Slides were visualized using a confocal microscope (Leica, DM IRE2) coupled to a spectral confocal system (Leica, TCS SP2) and micrographs captured.
2.8 Generation of PANDER-3-FLAG construct
The coding sequence of the pander gene was cloned upstream (5′) of the triple-FLAG repeat moiety of the pCMV-3FLAG-3a plasmid (Stratagene, 240197), employing EcoRV and XhoI restriction endonuclease sites within the multiple cloning region and construct integrity was confirmed by restriction digest and sequence analysis.
2.9 Immuno-fluorescent labeling of α–TC1-6 cells
α–TC1-6 cells nucleofected with pCMV-PANDER-3FLAG (described above) were cultured on glass coverslips (Fisherbrand®, 12-545-82-12CIR-1D) in complete DMEM. 48 hrs post-nucleofection, cells were fixed in 4% paraformaldehyde, then blocked using PSG: a solution of 0.01% saponin, 0.25% fish skin gelatin and 0.02% NaN3 in 1X PBS. Cells were then incubated in primary antibodies specific for FLAG and various sub-cellular markers using antibodies diluted in PSG for 1 hr at room temperature in a humidified environment. Coverslips were then rinsed three times with PSG at room temperature. Secondary antibodies in PSG were added to coverslips for 1 hr and cells again rinsed with PSG. Cells were then counterstained with DAPI and coverslips mounted using MOWIOL and stored at 4°C in the dark until visualized, and confocal micrographs captured.
2.10 PANDER secretion from α–cell lines
α–TC1-6 and α–TC6-Lenti-IR cells were transiently transfected with pShuttle-PANDER (as previously outlined). 24 hrs post-transfection, cells were rinsed and pre-incubated in DMEM, 3 mM glucose, 1% FBS for 1 hr prior to stimulation with L-Arginine (Sigma, A5006), Insulin (Sigma, I9278), or a combination of both at the indicated concentrations. For insulin stimulation experiments, cells were serum starved at 1% FBS for at least 5 hrs prior to stimulation. Where indicated, 200 nM wortmannin (Sigma, W1628) in DMSO was added to pre-incubation media prior to insulin stimulation. Post-stimulation, media was collected and cells lysed. Harvested media was concentrated by ammonium sulfate protein precipitation (0.55 g/ml media). Equal quantities of media or lysate protein were resolved by SDS-PAGE and probed for PANDER. Densitometric analysis was carried out as previously described and relative secreted PANDER determined as ratio of PANDER in media to that in lysate for each sample, and expressed as fold over negative control.
2.11 Hormone secretion
Glucagon secreted into culture media from α–TC1-6 cells was determined using Glucagon EIA (Alpco, 48-GLUHU-E01) or RadioImmunoAssay (Linco, GL-32K) kits according to manufacturer instructions and normalized to total protein content of cell lysates.
2.12 Data and statistical analyses
Statistical analyses were performed using the GraphPad Prism 5.0 software package. Statistical significance of differences was determined by Student’s t-test or analysis of variance (ANOVA) where more than two groups were compared. Threshold of statistical significance defined as P<0.05.