LEGENDS TO FIGURES IN APPENDIX
Appendix 1
Distribution of the genes differentially regulated in WAT of plin-/- mice. The right circle represents the transcripts identified with the Perfect-Match (PM)-only model. The left circle represents the transcripts identified with the Perfect-Match/Mismatch (PM/MM)-model. Numbers inside each portion of the circle are the genes that are differentially regulated between the plin-/- and the wild-type mice(dChip V.1.2).
Appendix 2
Quantitative real-time PCR of significant genes changed in the plin-/- mice. The data is plotted as a ratio of the expression of the selected gene transcript to that of a housekeeping gene (transferrin or cyclophilin) in the same sample, in the pooled total RNA from 3 plin-/- mice as compared to 3 wild-type littermates. The error bars represent 1 SD of 3 replicates. All of these are concordant with the microarray analysis as discussed in the text. CPT-2, Carnitine palmitoyl transferase-2; NADH dehydrogenase 1a, NADH dehydrogenase (ubiquinone) 1 9 (Ndufa9); ATP F1, ATP synthase, H+ transporting, F1 1 (Atp5c1); Succinate dehydrogenase, Succinate dehydrogenase complex B-Ip (SdhB); WT, wild type; KO, knock out
Appendix 4
Hierarchial clustering of WAT transcripts using dChip V 1.2. Scd1 and Scd2 were down-regulated in the same cluster in mouse plin-/-WAT, although in different sub-clusters. As shown, the first three columns represent wild type and the next three represent plin-/-samples with each column representing a separate experiment. The intensity of the colors represents the relative gene expression with bright red being highly expressed and dark blue representing very low expression.wt,wild type; ko,knock out; Scd,Stearoyl CoA desaturase
Appendix 5
Hierarchial clustering using dChip V 1.2ofScd1 and Scd2 transcripts in different tissues of plin-/- and wild-type mice. Scd1 and Scd2 cluster is depicted while clustering all the tissues and the genes. In each tissue, the first three columns represent wild type and the next three represent plin-/- samples with each column representing a separate experiment.In liver there was no change in Scd1 between wild type and plin-/-. Scd2 mRNA showed a high expression in WAT of wild type mice and a down-regulation in plin-/-. There is a low expression of Scd2 mRNA in liver, both in wild type and in plin-/-.L, liver; W,WAT; K,kidney; M,muscle; H,heart; Scd,Stearoyl CoA desaturase. The intensity of the colors represents the relative gene expression with bright red being highly expressed and dark blue representing very low expression.
Appendix 1
Appendix 2
Appendix 3
All the genes with a significant change in the plin-/- WAT are represented with the Affymetrix probe set numbers, GenBank accession numbers, gene annotations, gene symbol, fold changes, the model used in dChip for their detection and the functional category we assigned to the gene.
Microsoft Excel file attached separately.
Appendix 4
Appendix 5
Appendix 6
Functional annotation of transcripts for the Krebs cycle and Electron Transporters:
Krebs cycle genes up-regulated in plin-/- mice
1) Isocitrate dehydrogenase [NAD+] Idh3gdecarboxylates isocitrate into -ketoglutarate, and is activated by increasing ADP/ATP ratios and by Ca2+.
2) Succinate-CoA ligase GDP-forming Suclg1involved in phosphorylation.
3) Dihydrolipoamide succinyltransferase E2K (Dlst), component of the 2-oxoglutarate dehydrogenase complex that catalyzes the overall conversion of 2-oxoglutarate to succinyl-CoA and CO2, contains one covalently-bound lipoyl cofactor.
4) Dihydrolipoamide S-acetyltransferase (Pdc-e2), that catalyzes the overall conversion of pyruvate to acetyl-CoA and CO2, contains covalently-bound lipoyl cofactors and participates in the generation of acetyl groups from hydroxyethyl-thiamine pyrophosphate-e1.
5) Malate dehydrogenase (Mor1), catalyzes the interconversion of L-malate and oxaloacetate using nicotinamide adenine dinucleotide (NAD) as a coenzyme.
Krebs cycle genes down-regulated in plin-/- mice
1) Citrate synthase homologous, catalyzes the ligation of acetyl-CoA with oxaloacetate to yield citrate.
2) Isocitrate dehydrogenase 3 [NAD+] Idh3adecarboxylates isocitrate into -ketoglutarate.
3) Succinyl-CoA:3-ketoacid-CoA transferase (Oxct), key enzyme for ketone body catabolism, transfers the CoA moiety from succinate to acetoacetate, for the ketolytic energy production.
4) Succinate-CoA ligase ADP-forming Sucla2 involved in phosphorylation.
Electron transport chain genes up-regulated in plin-/- mice
1)NADH dehydrogenase (ubiquinone) 1 9 (Ndufa9), subunit of NADH-ubiquinone oxidoreductase (complex I), transfer of electrons from NADHto ubiquinone in the respiratory chain.
2) NADHdehydrogenase (ubiquinone) 1 9 (Ndufb9), subunit of NADH-ubiquinone oxidoreductase (complex I), transports electrons from NADH to ubiquinone in the respiratory chain.
3) NADHdehydrogenase (ubiquinone) 1 1 (Ndufa1), transcript increased in kidney that produces another subunit of complex I that codes for an essential component with a highly conserved two-domain structure which suggests that this feature is critical for its function and might act as an anchor for the NADH:ubiquinone oxidoreductase complex at the inner mitochondrial membrane, and is involved also in proton translocation.
4) Cytochrome c1 heme protein (Cyc1), the heme-containing component of the cytochrome b-c1 complex, accepts and transfers electrons to cytochrome C in the mitochondrial respiratory chain.
5) Succinate dehydrogenase complex, B-Ip (SdhB), nuclear gene encoding a mitochondrial protein for a subunit of complex II. SdhB transports electrons from succinate to ubiquinone.
6) Ubiquinol-cytochrome C reductase 1 (Uqcrc1), a core I respiratory protein, subunit of the ubiquinol-cytochrome C oxidoreductase (complex III). Uqcrc1 may mediate formation of the complex between cytochromes C and C1.
7)Cytochrome C oxidase VIIa 1 (Cox7a1) is also one of the nuclear-coded polypeptide chains of cytochrome C oxidase, the terminal oxidase in mitochondrial electron transport.
8) Cytochrome C oxidase polypeptide VIII (Cox8b) is other of the nuclear-coded polypeptide chains of cytochrome C oxidase, the terminal oxidase in mitochondrial electron transport, and catalyzes the transfer of reducing equivalents from cytochrome C to molecular oxygen and pumps protons across the inner mitochondrial membrane.
9) Cytochrome C oxidase assembly protein 2 (Sco2), also a nuclear gene for a mitochondrial product. Sco2 act as a copper chaperone, transporting copper to the Cu(a) site on the cytochrome C oxidase subunit II (ref. a).
10) ATP synthase, H+ transporting, F1 1 (Atp5a1), is a regulatory subunit in the production of ATP from ADP in the presence of a proton gradient across the membrane.
11)ATP synthase, H+ transporting, F1 1 (Atp5c1), produces ATP from ADP in the presence of a proton gradient across the membrane, is important in regulating ATPase activity and the flow of protons through the CF(0) complex.
12)ATP synthase, H+ transporting, F0 2 (Atp5c2), this protein is one of the chains of the nonenzymatic membrane component (F0) of mitochondrial ATPase.
Electron Transport Chain down-regulation in plin-/- mice
In muscle, a transcript for NADH-ubiquinone oxidoreductase homologous (Ndufs1), a subunit of complex 1, have a decreased expression.
Electron transport NADPH dependent gene transcripts increased in plin-/- mice
The transcript of cytochrome b-245 alpha polypeptide was up-regulated in WATand to a lesser degree in liver, whereas cytochrome P450 2b10 phenobarbitol inducible (Cyp2b10) was down-regulated only in liver.
1) Cytochrome b-245 polypeptide (Cyba), a membrane bound superoxide generator that belongs to the p22phox family.
2) Neutrophil cytosolic factor 4, p40phox (Ncf4), through interaction with an SH3 domain is responsible for the down-regulation of NADH-oxidase, and associates primarily with p67phox to form a complex with p47phox.
3) NADPH dehydrogenase quinone 1 (Nqo1), quinone reductase connected with conjugation reactions of hydroquinones.
4) Flavin containing monooxygenase 5 (Fmo5). Synthetic progestin induced messages for fmo5 in cancer cell lines stably and independently expressing progesterone B-receptors (ref. b).
5) Cytochrome P450 2b10 (Cyp2b10), enzyme involved in an NADPH-dependent electron transport pathway, oxidizes a variety of structurally unrelated compounds, including steroids, fatty acids, and xenobiotics. In plin-/- liver this gene was decreased. Lipopolysaccharides (0.3mg/kg) suppressed Cyp2b10 mRNA 12 hr. after injection in wild-type mice (ref. c).
Referencesfor Appendix 6
a) Salviati L, Hernandez-Rosa E, Walker WF, Sacconi S, DiMauro S, Schon EA, Davidson MM: Copper supplementation restores cytochrome C oxidase activity in cultured cells from patients with Sco2 mutations. Biochem J363:321-327, 2002
b) Miller MM, James RA, Richer JK, Gordon DF, Wood WM, Horwitz KB: Progesterone regulated expression of flavin-containing monooxygenase 5 by the B-isoform of progesterone receptors: implications for tamoxifen carcinogenicity. J Clin Endocrinol Metab 82:2956-2961, 1997
c) Li-Masters T, Morgan ET: Down-regulation of phenobarbital-induced cytochrome P4502B mRNAs and proteins by endotoxin in mice: independence from nitric oxide production by inducible nitric oxide synthase. Biochem Pharmacol64:1703-1711, 2002
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