MK5 Microarray Data
Microarrays were performed to compare the transcription pattern in cells expressing constitutive active MK5 with control cells. For this purpose, we generated PC12 cells that were stably transfected with an expression plasmid for the constitutive active MK5 L337A mutant. This plasmid contains the tetracycline off (tet-off) inducible promoter, allowing MK5 L337A expression after withdrawal of doxocyclin. RNA was purified from cells grown in the presence of doxocycline (no expression of the constitutive active MK5) or in the absence of doxocycline (expression of active MK5) and used in the microarray analyses.
The plasmid allowing doxocycline-inducible expression of the active MK5 mutant was generated as follws. By site-directed mutagenesis, we mutated the MK5 cDNA sequence of the plasmid pcDNA-HA-MK5_WT plasmid into pcDNA-HA-MK5 L337A using the primers 5'-CCC-AAG-CTT-GAC-GCG-TCC-ATG-TAT-GAT-G-3' (and the reversed complement). This mutation changes amino acid residue 337 from leucine into alanine. This mutation renders MK5 into a constitutive active mutant [Ole Morten Seternes, Bjarne Johansen, Beate Hegge, Mona Johannessen, Stephen M. Keyse, and Ugo Moens. Both binding and activation of p38 MAPK play essential roles in regulation of nucleocytoplasmic distribution of MAPK activated protein kinase 5 by cellular stress. Molecular and cellular biology, 22(20):6931-6945,2002]. The mutation in pcDNA-HA-MK5 L337A plasmid was verified by sequencing and the plasmid was then digested with MluI/NotI and cloned into the corresponding sites of pTRE2 to generate the plasmid pTRE2-MK5-L337A. PC12 cells were transfected and stable cell lines were isolated by selected on hygromycin.
Two 6-well plates with 5.10^5 PC12 TetOff cells (BD Biosciences) were transfected with 14 microg of pTRE2-MK5_L337A and 2 microg pTKHyg per well using lipofectamine 2000 (Invitrogen) [Luigi Pianese, Luca Busino, Irene De Biase, Tiziana de Cristofaro, Maria S. Lo Casale, Paola Giuliano, Antonella Monticelli, Mimmo Turano, Chiara Criscuolo, Alessandro Filla, Stelio Varrone, and Sergio Cocozza. Up-regulation of c-Jun N-terminal kinase pathway in Friedreich's ataxia cells. Human Molecular Genetics, 11(23):2989-2996, 2002] After 3.5h, the medium was changed and supplemented with 10 ng/ml Doxycycline (Sigma). 24h after transfection, cells were transferred to 10 cm dishes with fresh medium and Doxycycline. 48h after transfection, 100 microgram/ml of Geneticin (Gibco) and 200 microg/ml Hygromycin B (Invitrogen) was supplied additionally to the medium. The cells were grown until visible colonies of resistant cells could be detected. From each plate two colonies were transferred in threefold dilution to a $ 96$ well plate. For positive clones, we confirmed the transgene expression though reverse transcriptase-PCR and western blot. Cells were maintained in DMEM supplied with 10% horse serum and 5% fetal bovine serum, 2mM L-glutamine, penicillin (110 units/ml) and streptomycin (100 microgram/ml). Additionally, 50 microgram/ml of Geneticin, 100 microgram/ml Hygromycin B were supplied to maintain selection. To suppress HA-MK5_L337A expression during ordinary cell culture, we added 10ng/ml Doxycycline. Total RNA was isolated from non-induced and induced cells and converted into cDNA by reverse transcriptase (Iscript, BioRad).
Subsequently, we hybridized 4 microarray slides, two containing cDNA from an induced (Cy3) sample and two containing an uninduced sample (Cy5). Samples were labeled with the 3DNA 350S HS labeling kit (Genisphere). The slide layout was a KTH Rat27K Oligo microarry Operon v3.0. A Tecan HS 4800 scanned the microarrays and the images were analyzed using the Genepix 4000B (Molecular Devices), which reported a constant gain of 950/800. We obtained more than 70% hybridization (measured as #spots > median + 1SD). Spots with too large an intensity (> 90% of the maximum) or too large a regulation (> x10) were removed. The data was quantile normalized after which we modelled the probe measurement errors/efficiency according to [Werner Van Belle, Nancy Gerits, Kirsti Jakobsen, Vigdis Brox, Marijke Van Ghelue, Ugo Moens; Confidence Intervals on Microarray Measurement of Differentially Expressed Genes: A Case Study on the effects of MK5, TAF4 and FKRP on the transcriptome; Gene Regulation and Systems Biology; Libertas Academus Press; nr 1; pages 57-72; May 2007] and filtered out those regulations for which the 95% confidence interval was inconclusive.
The data can be freely used. We would however appreciate referring to Werner Van Belle, Nancy Gerits, Kirsti Jakobsen, Vigdis Brox, Marijke Van Ghelue, Ugo Moens; Confidence Intervals on Microarray Measurement of Differentially Expressed Genes: A Case Study on the effects of MK5, TAF4 and FKRP on the transcriptome; Gene Regulation and Systems Biology; Libertas Academus Press; nr 1; pages 57-72; May 2007. If interested in MK5 you can contact Ugo Moens (firstname.lastname@example.org, email@example.com) or Nancy Gerits (firstname.lastname@example.org) directly. If interested in the analysis process please contact Werner Van Belle (email@example.com)