We have reported that retinoids both RAs and

We have reported that retinoids, both RAs and retinoic serine protease inhibitors receptor (RAR)/retinoid X receptor (RXR) analogs, are capable of enhancing macrophage cholesterol efflux, and these events involve activation of the LXR pathway [1,2]. Specifically, retinoid mediated macrophage cholesterol efflux has been shown to be influenced by the LXR regulated genes, sterol regulatory element-binding protein 1c (SREBP-1c) and ABCA1. To obtain further insights into the LXR regulatory events, RAW 264.7 macrophages were transfected with an LXRE reporter plasmid (pLXREx3-Luc; [1,3]), and the effects of selective analogs with affinities to both RAR (TTNPB; Sigma-Aldrich; St. Louis, MO) and RXR (SR11233; Sigma-Aldrich) on luciferase/LXR activity were determined. As illustrated in Fig. 2, LXRE transfected macrophages treated with TTNPB (5µM; [1]) and SR11233 (5µM; [1]), individually, resulted in 2.6±0.4 and 3.2±0.6 fold increases in luciferase activity over untreated cells, respectively. LXR activity was unaffected in response to 0.1mM (Bu)2cAMP. Addition of (Bu)2cAMP (0.1mM) to either TTNPB or SR11233 incubation significant enhanced (p<0.001) LXR activity, when compared with controls. Co-incubation of SR11233 and (Bu)2cAMP displayed ~2 fold increase in LXR activity, over the combined response seen with TTNPB and (Bu)2cAMP. These data are connected with our previous findings that demonstrate that both RAs and RAR/RXR analogs effectively enhance macrophage cholesterol efflux through activation of the LXR pathway [1,2].
Experimental design, materials and methods
Acknowledgments
Data These data were obtained as described in detail in [1]. SAXD diffractograms, SAXD and WAXD spacings, 31P NMR and 2H NMR are included obtained from idebenone-DPPC and idebenol/DPPC samples.
Experimental design, materials and methods Materials. 1-Palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC); 1, 2-dipalmitoyl-sn-phosphatidylcholine (DPPC) and 1, 2-d62-sn- dipalmitoyl-sn-phosphatidylcholine (DPPC-d62) were obtained from Avanti Polar Lipids (Birmingham, Alabama, USA). Idebenone was from TCI Europe N.W. (Zwijndrecht, Belgium) and all other chemicals were highly pure from Sigma Chemical Co. (Madrid, Spain).
Methods
Acknowledgments This work was supported partially by funds from Universidad de Murcia (Grant 368).
Data In the present work related to [1], we show differences in amounts of individual forms of respiratory chain complexes I, III and IV quantified from western blots of 2D BNE/SDS PAGE analysis, as determined in mitochondria of SURF1 and SURF1 mouse fibroblasts and tissues (heart, muscle, brain, liver) and also in mitochondria of human control and SURF1 deficient fibroblasts (Figs. 1–3). Then we show data (Fig. 4) from analysis of fibroblast cell lines from SURF1mouse, SURF1 patient and controls, in which translation of mitochondrial DNA encoded proteins was reversibly inhibited with doxycycline (DOX). After DOX removal, the formation of newly synthetized COX in time (0–96h) was assessed by SDS PAGE and western blot analysis.
Experimental design, materials and methods
Data
Experimental design, materials and methods
RNA extraction
The cDNA library construction and sequencing The TruSeq RNA Sample Prep Kit (Illumina) was used to isolate mRNA from about 5μg of total RNA using oligo-d(T)25 magnetic beads. The mRNA was sheared with ions into ~200nt fragments and was reverse-transcribed into cDNA. We then used the TruSeq PE Cluster Kit v3 (Illumina) to perform end repair, add an ‘A’-base to the blunt ends, and ligate the cDNA to paired-end adapters. The cDNA samples were amplified through 15 cycles of PCR. The amplification products were electrophoresed on a 2% Certified Low Range Ultra Agarose gel (Bio-Rad) and purified according to appropriate size of DNA fragments suitable for Illumina sequencing. The purified products were quantified using PicoGreen (Life Technologies) and a TBS-380 Mini-Fluorometer (Promega) and loaded on an Illumina cBot system for cluster generation by bridge PCR amplification. Sequencing was performed on an Illumina HiSeq 2500 platform.