a template, and forward: 59-ggaattc- ccgcgg ATGCCCAAGAA- GAAGAGGAAGGTGTC, reverse (59-gaattccttaaggagctc GAA- CAAACGACCCAACACCCGTG) primers. After digesting with SacII and AflII, the PCR product was inserted downstream of human INS promoter in phINS-EGFP that was digested with SacII and AflII and removed EGFP-pA by gel extraction, generating plasmid phINS-Cre-pA. To combine phINS-Cre-pA with pCMV- L-mCherry-pA-L-EGFP-pA, phINS-Cre-pA fragment was ob- tained from plasmid phINS-Cre-pA by digesting with SalI plus AflII and blunted at both sites with Klenow. The fragment was inserted in the reverse orientation (overwhelmingly, but not in the right orientation) upstream of CMV promoter in pCMV-L- mCherry-pA-L-EGFP-pA digested with AseI and blunted at both sites with Klenow. DNA sequences were confirmed using standard sequencing protocols (USC DNA Sequencing Core Facility). Electrophysiology of HIT T15 cells Cells were patch-clamped 2–4 days after plating. Current recordings were obtained in conventional whole cell patch-clamp configuration with an Olympus IX70 inverted microscope, EPC-9 amplifier, and Pulse software (HEKA Electronics). The glass- bottomed chambers with adherent cells were washed with PBS and then filled with standard extracellular solution consisting of 140 mM NaCl, 2.8 mM KCl, 10 mM HEPES, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose, pH adjusted to 7.2–7.4, and osmolarity adjusted to 300–310. The pipette solutions contained 10 mM NaCl, 145 mM cesium glutamate, 10 mM HEPES, 2 mM CaCl2, 1 mM MgCl2, 0.1 mM EGTA, 2 mM ATP, 0.3 mM GTP, titrated with 5 M CsOH to pH 7.2–7.4, and osmolarity adjusted to 290–300. Measurement of [Ca2+]i in response to glucose stimulation In order to observe calcium changes associated with glucose stimulation, we used the ratiometric calcium dye Fura2-AM (Invitrogen). The intracellular Ca2+ concentration was determined by the ratio of the excitation of the ratiometric calcium dye Fura2- AM (Invitrogen) at 350 and 380 nm using the Polychrom V by Till Photonics. Emission was measured at 500–520 nm using a Photometric Cascade CCD camera and Metafluor Software. References 1. Efrat S, Linde S, Kofod H, Spector D, Delannoy M, et al. (1988) Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene. Proc Natl Acad Sci U S A 85: 9037–9041. 2. Gilligan A, Jewett L, Simon D, Damjanov I, Matschinsky FM, et al. (1989) Functional pancreatic beta-cell line from SV40 T-antigen transgenic mouse. Diabetes 38: 1056–1062. 3. Radvanyi F, Christgau S, Baekkeskov S, Jolicoeur C, Hanahan D (1993) Pancreatic beta cells cultured from individual preneoplastic foci in a multistage tumorigenesis pathway: a potentially general technique for isolating physiolog- ically representative cell lines. Mol Cell Biol 13: 4223–4232. 4. Skelin M, Rupnik M, Cencic A (2010) Pancreatic beta cell lines and their applications in diabetes mellitus research. ALTEX 27: 105–113. 5. Ulrich AB, Schmied BM, Standop J, Schneider MB, Pour PM (2002) Pancreatic cell lines: a review. Pancreas 24: 111–120. 6. Hasegawa K, Nakatsuji N (2002) Insulators prevent transcriptional interference between two promoters in a double gene construct for transgenesis. FEBS Lett 520: 47–52. 7. Santerre RF, Cook RA, Crisel RM, Sharp JD, Schmidt RJ, et al. (1981) Insulin synthesis in a clonal cell line of simian virus 40-transformed hamster pancreatic beta cells. Proc Natl Acad Sci U S A 78: 4339–4343. 8. Fromont-Racine M, Bucchini D, Madsen O, Desbois P, Linde S, et al. (1990) Effect of 59-flanking sequence deletions on expression of the human insulin gene in transgenic mice. Mol Endocrinol 4: 669–677. 9. Laimins L, Holmgren-Konig M, Khoury G (1986) Transcriptional ‘‘silencer’’ element in rat repetitive sequences associated with the rat insulin 1 gene locus. Proc Natl Acad Sci U S A 83: 3151–3155. 10. Hay CW, Docherty K (2006) Comparative analysis of insulin gene promoters: implications for diabetes research. Diabetes 55: 3201–3213. A Dual-Color Reporter for Insulin+ Beta Cells Cells were incubated at room temperature for 1 hour in standard patch-clamp external solution with a modified glucose concentration of 3 mM and supplemented with 4 mM Fura2-AM. The cells were washed three times with 3 mM glucose external solution before recording. Fura-2 fluorescence was detected with a timelapse of 300 ms at 500–520 nm wavelength following excitation at 350 nm (F350) and 380 nm (F380). The ratio, F350/F380, was calculated after subtracting background from each wavelength using Igor programming software. After recording fluorescence measurements in 3 mM glucose, high- glucose extracellular solution was added for a final concentration of 14 mM glucose to stimulate glucose response. The ratio, F350/ F380, was again obtained using Igor software. Supporting Information Figure S1 Transfection efficiency in MB-231 and HIT- T15 cells by pEGFP-N1. (DOC) Figure S2 Transiently transfected HIT-T15 and beta- TC-6 cells with the indirect dual-color reporter. (a, b) Confocal images of HIT-T15 (a) and beta-TC-6 (b) cells. Green and red fluorescence is colocalized in one cell. (DOC) Acknowledgments The authors thank S. Wang, L. Zhang, D. Gibson, and Le Ma (USC) for reagents, and C. Newgard (Duke University) for INS-1 832/13 cells. The authors also thank other members of the Chow lab for assistance with using equipment. Author Contributions Analyzed the data: NSL. Contributed to very helpful discussions/advice: PMS KH MP AW RHC. Edited the manuscript: PMS KH MP AW RHC. Executed experiments of constructing reporters with SK, and all other molecular and cell biological assays: NSL. Performed electrophysiological experiments: JGR. Contributed to measurement of [Ca2+]I: MZ. Contributed to imaging of [Ca2+]I: AW. Contributed pINS-GFP construct: KH. Helped write the manuscript: PMS. Designed and wrote the manuscript: NSL RHC. 11. Le Lay J, Matsuoka TA, Henderson E, Stein R (2004) Identification of a novel PDX-1 binding site in the human insulin gene enhancer. J Biol Chem 279: 22228–22235. 12. Itier JM, Douhet P, Desbois P, Joshi RL, Dandoy-Dron F, et al. (1996) Human insulin gene expression in transgenic mice: mutational analysis of the regulatory region. Differentiation 60: 309–316. 13. Alwine JC (1985) Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins. Mol Cell Biol 5: 1034–1042. 14. 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Hohmeier HE, Mulder H, Chen G, Henkel-Rieger R, Prentki M, et al. (2000) Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel- dependent and -independent glucose-stimulated insulin secretion. Diabetes 49: 424–430. 20. Swope SL, Schonbrunn A (1984) Bombesin stimulates insulin secretion by a pancreatic islet cell line. Proc Natl Acad Sci U S A 81: 1822–1826. PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e35521