ABSTRACT: Cell culture and establishment of cell lines The mouse pancreatic beta cell line MIN6 at passage 24 was obtained with permission from Dr. Jun-ichi Miyazaki at Osaka University (Miyazaki et al., 1990). MIN6 cells were cultured as described previously (Miyazaki et al., 1990). Two independent Sirt1-overexpressing MIN6 cell lines (MIN6-Sir2OE1 and Sir2OE2) were established by infection with the ecotropic pBabe-Sirt1 retrovirus carrying the mouse Sirt1 gene and a neomycin-resistant gene (Revollo et al., 2004) and selection in the presence of 280 micrograms/ml of G418 (Invitrogen). Two control cell lines (MIN6-Neo1 and Neo2) were also established by infection with the pBabe-neo control retrovirus. To establish Sirt1-knockdown MIN6 cell lines, small interfering RNAs (siRNAs) were designed against the Sirt1 gene, and each double-stranded siRNA template was cloned in the mU6pro vector (Yu et al., 2002, a gift from Dr. David L. Turner at University of Michigan, Ann Arbor). Sirt1 siRNA sequences are available upon request. Two independent Sirt1-knockdown MIN6 cell lines (MIN6-Sir2KD1 and KD2) were established by co-transfection with expression vectors carrying two different siRNAs and a neomycin-resistant marker plasmid and selection in the presence of 400 micrograms/ml G418. A control cell line (MIN6-mU6pro) was also established using the mU6pro vector. All experimental and control cell lines were propagated carefully to ensure equivalent passage numbers and maintain healthy cellular morphology. Microarray hybridizations and data collection All cell lines were cultured in media containing 5 mM glucose for four days prior to RNA collection. RNA samples were purified from two Sirt1-overexpressing (Sir2OE1 and Sir2OE2), two Sirt1-knockdown (Sir2KD1 and Sir2KD2), and three control (Neo1, Neo2 and mU6pro) MIN6 cell lines using Trizol (Sigma) according to the manufacturer’s protocol. The quality of RNA was examined by capillary electrophoresis and denaturing agarose gel electrophoresis. RNA from MIN6-Neo1 and Neo2 was combined prior to cDNA synthesis. Eight micrograms of total RNA from each sample were converted to cDNA by using the 3DNA Array 350 Expression Array Detection Kit (Genisphere, PA) according to the manufacturer’s protocol. Microarray hybridization was then conducted with Cy3- and Cy5-labeled dendrimers as described (Scearce et al., 2002) with the following modifications. Hybridization was conducted at 42ºC for 18 h in MWG Coverslips Hybridization Buffer (MWG USA, NC), and all post-hybridization washes were carried out at 25ºC. To increase the accuracy of the microarray analysis, we performed dye-swap experiments for each pair of cell lines (Sir2OE1 vs. Neo1+Neo2, Sir2OE2 vs. Neo1+Neo2, Sir2KD1 vs. mU6pro, and Sir2KD2 vs. mU6pro). Microarray hybridizations were performed in duplicate for each pair with the exception that the Cy3-Cy5 labeling scheme was swapped between hybridizations, i.e., (control-Cy3, experimental-Cy5) and (experimental-Cy5, control-Cy3). Including dye-swaps, four microarray slides were used for each pair-wise comparison. A ScanArray Express HT scanner and accompanying software (PerkinElmer, MA) was used to scan the slides and analyze the raw data, including normalization according to the Lowess method (Yang et al., 2002). Microarray analysis Spots used for statistical analysis satisfied the following criteria on at least three of the slides for both sets of comparisons: (1) ScanArray Express flag = 3, (2) Signal to noise ratio (defined as (mean foreground - mean background)/(standard deviation of background)) ≥ 2 in both channels. Spot-specific dye bias was calculated as the arithmetic mean of the log2 transform of the Lowess-normalized ratio of the medians (Yang et al., 2002) across dye-swapped pairs of hybridizations for the given set of experiments (either Sirt1 overexpression or knockdown). The spot-specific dye bias was subtracted from the log2 transform of the Lowess-normalized median of ratios of the foreground pixels in the Cy5 and Cy3 channels to obtain the log2 transform of the “expression ratio”. For up-regulated genes, fold changes are equivalent to the expression ratio. For down-regulated genes, fold changes are the negative inverse of the expression ratio. In either case, fold change = ±2^|log2(expression ratio)|. To reduce the effect of multiple hypothesis testing, significant changes were demarcated by a difference of least 3 standard deviations (99.74% of normally distributed data) from the control. In addition, only genes that differed by at least 1.8-fold from the control were considered. References Miyazaki, J.-I., Araki, K., Yamato, E., Ikegami, H., Asano, T., Shibasaki, Y., Oka, Y., and Yamamura, K. (1990). Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology 127, 126-132. Revollo, J. R., Grimm, A. A., and Imai, S. (2004). The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells. J Biol Chem 279, 50754-50763. Scearce, L. M., Brestelli, J. E., McWeeney, S. K., Lee, C. S., Mazzarelli, J., Pinney, D. F., Pizaro, A., Stoeckert Jr., C. J., Clifton, S. W., Permutt, M. A., et al. (2002). Functional genomics of the endocrine pancreas: The pancreas clone set and PancChip, new resources for diabetes research. Diabetes 51, 1997-2004. Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., Ngai, J., and Speed, T. P. (2002). Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30, e15. Yu, J.-Y., DeRuiter, S. L., and Turner, D. L. (2002). RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA 99, 6047-6052. Keywords = Sir2 Keywords = Sirt1 Keywords = Sir2[alpha] Keywords = NAD-dependent deacetylase Keywords = pancreatic ß cells Keywords = BESTO Keywords = beta cell-specific Sirt1-overexpressing transgenic mice Keywords = transgenic Keywords = glucose tolerance Keywords = insulin secretion Keywords = microarray Keywords = gene expression profiling Keywords = Ucp2 Keywords = ATP Keywords = glucose sensitivity Keywords = aging Keywords = diabetes Keywords: ordered
