Transcriptional Regulation of Pro-apoptotic Protein Kinase C-delta: Implications for Oxidative Stress-induced Neuronal Cell Death

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1 SUPPLEMENTAL DATA Transcriptional Regulation of Pro-apoptotic Protein Kinase C-delta: Implications for Oxidative Stress-induced Neuronal Cell Death Huajun Jin 1, Arthi Kanthasamy 1, Vellareddy Anantharam 1, Ajay Rana 2 and Anumantha G. Kanthasamy 1 1 Parkinson s Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Department of Biomedical Sciences, Iowa State University, Ames, Iowa USA 2 Department of Pharmacology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, USA Experimental Procedures Quantitative real-time RT-PCR- Total RNA was isolated from fresh cell pellets using the Absolutely RNA Miniprep Kit (Stratagene, La Jolla, CA) according the manufacturer s protocols. Aliquots of 3 μg of total RNA were used for first strand cdna synthesis by random primer and AffinityScript Multiple Temperature Reverse Transcriptase in a 20 μl reaction volume using an AffinityScript QPCR cdna Synthesis Kit (Stratagene). Quantitative RT-PCR was performed in an Mx3000P QPCR System (Stratagene) using the Brilliant SYBR Green QPCR Master Mix Kit (Stratagene), with cdnas corresponding to 150 ng of total RNA, 12.5 μl of 2 master mix, μl of reference dye, and 0.2 μm of each primer in a 25-μl final reaction volume. All reactions were performed in triplicate. Sequences for PKCδ primers used in this study are shown in Table S1. β-actin was used as internal standard with the primer set purchased from Qiagen (QuantiTect Primers, catalog number QT ). The PCR Cycling conditions contained an initial denaturation at 95 C for 10 min, followed by 40 cycles of denaturation at 95 C for 30 sec, annealing at 60 C for 30 sec, and extension at 72 C for 30 sec. Fluorescence was detected during the annealing step of each cycle. Dissociation curves were run to verify the singularity of the PCR product. The data

2 were analyzed using the comparative threshold cycle (Ct) method. The PKCδ mrna values were normalized to the amount of β-actin internal control in each sample and expressed as the fold of mrna levels of control samples (set to 1). Methylation specific PCR (MSP)- For MSP experiments, genomic DNA was isolated using the DNeasy blood & tissue kit (Qiagen). Bisulfite modification was subsequently carried out on 500 ng of genomic DNA by the MethylDetector bisulfite modification kit (Active Motif, Carlsbad, CA) according to the manufacturer s instructions. Two pairs of primers were designed to amplify specifically methylated or unmethylated PKCδ sequence using MethPrimer software (1). The cycling condition was: 94 C for 3 min, after which 35 cycles of 94 C for 30 sec, 54 C for 30 sec, 68 C for 30 sec, and finally 72 C for 5 min. PCR products were loaded onto 2% agarose gels for analysis. Statistical analysis- Statistical analyses were performed using the GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA). One-way analysis of variance (ANOVA test) followed by the Tukey multiple comparison tests was used for statistical comparisons, and differences were considered significant if P-values less than 0.05 were obtained.

Fig. S1. Additive activation of PKCδ promoter transcription by Sp2 and Sp3. The expression plasmids pn3-sp3 FL, pcdna-sp2, and empty vector pn3 or pcdna3.

the bar graph. Luciferase activity was determined after 24 h of transfection. Transfection efficiency was normalized by β-galactosidase activity.

3 Fig. S1. Additive activation of PKCδ promoter transcription by Sp2 and Sp3. The expression plasmids pn3-sp3 FL, pcdna-sp2, and empty vector pn3 or pcdna3.1 were cotransfected along with the PKCδ promoter reporter construct pgl3-147/+289 into NIE115 either alone or in the different combinations, as indicated below the bar graph. Luciferase activity was determined after 24 h of transfection. Transfection efficiency was normalized by β-galactosidase activity. Data expressed as fold induction relative to that obtained from cells transfected with empty vector alone and represent the mean ± SEM of three independent experiments performed in triplicate.

Fig. S2. Treatment with methylation inhibitor 5 -aza-2 -deoxycytidine (5-Aza-dC) significantly increased endogenous PKCδ mrna and attenuated PKCδ promoter methylation in NIE115 cells.

4 Fig. S2. Treatment with methylation inhibitor 5 -aza-2 -deoxycytidine (5-Aza-dC) significantly increased endogenous PKCδ mrna and attenuated PKCδ promoter methylation in NIE115 cells. NIE115 cells were treated with varying doses of 5-Aza-dC for 24 h, as indicated, and cells were than collected for real-time PCR analysis of PKCδ mrna (A) or bisulfate-modification and subsequent MSP analysis (B) with primers for methylated (M) and unmethylated (U) DNA. PCR bands in (B) were analyzed using the one-dimensional image analysis software (Kodak Molecular Imaging System), and the relative methylation status was expressed as ratio of methylated versus unmethylated. (*, p<0.05; between the control and 5-Aza-dC-treated samples)

5 Table S1: List of primer sequences used in the study. Primer Sequence (5-3 ) P-1694 F GTCTATCTCGAGGATCTGACGCCCTCTTCTGGAGT P-1193 R1 GTCCTGATAACTGTCCCCACCCCAT P-1217 F ATGGGGTGGGGACAGTTATCAGGAC P+289 R GTCTATAAGCTTACCTCACCCAGGTGCCGG P-1192F ATATATCTCGAGTGGGGACTTAAATACTAATT P-1193R2 ATATATAAGCTTGTCCTGATAACTGTCCCCAC P-660F1 ATATATCTCGAGTATCCTCCCAGGAAGAGTTCTCG P-660F2 P-659R ATATATGGTACCTATCCTCCCAGGAAGAGTTCTCG ATATATAAGCTTTACAAGAGGGTTCTAATAGCC P-147 F ATATATCTCGAGTCTCGGGCAGGACTGGAACC P-148R ATATATAAGCTTGAAGGAGCTGGGAGGTCTCC P+2 F P+2R ATATATCTCGAGTCCTGGGCTCCATTGTGTGTG GTCTATAAGCTTAGGCACCGACGGGGCTTCC P-1072F ATATATCTCGAGCCCCAATGTACATTTAAAATAAGG P-882F ATATATCTCGAGGATCTCGTTAAGGATGGTTGTG P-822F ATATATCTCGAGTCGGAAGAGCAGTCGGGTGCTC P-712F ATATATCTCGAGAGGTAGTTTTCCAGAAGGAAC P-560F ATATATCTCGAGGAGCACTGGAGTATTATTCTGAG P-460F ATATATCTCGAGAGCCCAGGAAGTCATTTCTTTG P-371F ATATATCTCGAGATTTGGTGCTCAGACTTTGGGC P-300F ATATATCTCGAGTCTTATGAGCTTGACTGAGCAAGG P-250F ATATATCTCGAGAGACAGTGAGATGGGGGCAGA P-197F ATATATCTCGAGTGAGACAAACTGGCTAGAACCTC P-561R1 P-561+2F ATATATGCTAGCAGGGGGAGAAAGCAGGAGAAT TGCTTTCTCCCCCTCCTGGGCTCCATTGTGTGTG P-561+2R P+209R CAATGGAGCCCAGGAGGGGGAGAAAGCAGGAGAA GTCTATAAGCTTACGTGAGCTGGGGGTCCAGC P+165F mgc(1) F mgc(1) R mgc(2) F mgc(2) R mgc(3) F mgc(3) R mgc(4) F mgc(4) R mcaccc F mcaccc R ATATATCTCGAGTTGCAACTCAAAGAGGCTGA GGACCCCCAGCTCACGTAAGCTTAGCTTCGAAG ACGTGAGCTGGGGGTCCAGCGCGTCTCAGC TGGGCGGAGCTTCGAAGAAGCTTGCGCCCGTGG CTTCGAAGCTCCGCCCACGTGAGCTGGGGG AGGGGCGGGCGCCCGTGAAGCTTGTCCTGAGTG CACGGGCGCCCGCCCCTTCGAAGCTCCGCC GGGCGGGTCCTGAGTGGAAGCTTGACCGGGGCC CCACTCAGGACCCGCCCCACGGGCGCCCGC GTGTGCAGTGCTCAACTCTAACCTTTAACTTGGCCT GTTGAGCACTGCACACACAATGGAGCCCAG

6 Table S1 (continued). Primer Sequence (5-3 ) Amplicon mgc(2,3) F AGAAGCTTGCGCCCGTGAAGCTTGTCCTGAGTG mgc(2,3) R CACGGGCGCAAGCTTCTTCGAAGCTCCGCC mgc(1,2,3) R PKCδ Fq PKCδ Rq ChIP F=P+2F ChIP R=P+289R Methylated F Methylated R Unmethylated F Unmethylated R CACGGGCGCAAGCTTCTTCGAAGCTAAGCT TCTGGGAGTGACATCCTAGACAACAACGGG CAGATGATCTCAGCTGCATAAAACGTAGCC ATATATCTCGAGTCCTGGGCTCCATTGTGTGTG GTCTATAAGCTTACCTCACCCAGGTGCCGG TGTAATTTAAAGAGGTTGAGACGC TAACCGTCTCTAACTCTTATAACGC TAGTTGGTTAGTGGGGAGTTTTG TTAACCATCTCTAACTCTTATAACACC F, Forward; R, Reverse; q, quantitative RT-PCR; m, mutant primers; ChIP, primers used for ChIP experiments; Methylated and unmethylated, primers used for MSP experiments. Sequence of primers for constructions mouse PKCδ promoter reporter plasmids, site-directed mutagenesis, real-time RT-PCR, and ChIP experiments. Boldface letters indicate mutated bases.

7 Table S2. Sense sequences of the oligonucleotides used in EMSAs. Probe/Competitor Sense oligonucleotide (5-3 ) PkcδGC(1, 2) PkcδGC(1, 2) mutant Sp1 consensus Sp1 consensus mutant PkcδGC(1) PkcδGC(2) CACGTGGGCGGAGCTTCGAAGGGGCGGGCGCC CACGTaaTCttAGCTTCGAAGaaTCTttCGCC ATTCGATCGGGGCGGGGCGAGC ATTCGATCGaaTCtttGCGAGC GCTCACGTGGGCGGAGCTTC CTTCGAAGGGGCGGGCGCCCG Nucleotide sequences of the consensus binding motif are underlined. Mutated base pairs in mutant oligos are highlighted in bold and in lowercase. References 1. Li, L. C., and Dahiya, R. (2002) Bioinformatics (Oxford, England) 18(11),

Technical Review. Real time PCR

Technical Review. Real time PCR Technical Review Real time PCR Normal PCR: Analyze with agarose gel Normal PCR vs Real time PCR Real-time PCR, also known as quantitative PCR (qpcr) or kinetic PCR Key feature: Used to amplify and simultaneously