SUPPLEMENTAL METHODS Cell culture RNA extraction and analysis Immunohistochemical analysis and laser capture microdissection (LCM)
|
|
|
- Marvin Hubbard
- 5 years ago
- Views:
Transcription
1 SUPPLEMENTAL METHODS Cell culture Human peripheral blood mononuclear cells were isolated from healthy donors by Ficoll density gradient centrifugation. Monocyte differentiation to resting macrophages () was induced by 6 days of culture in RPMI 60 medium (Invitrogen, France) supplemented with gentamicin (0 µg/ml), L-glutamine ( mmol/l) (Sigma-Aldrich, France) and 0% pooled human serum (Abcys, France). Alternatively differentiated macrophages (M) were obtained by stimulating monocytes with recombinant human IL- ( ng/ml) (Promocell, Germany) for 6 days. Classical differentiated (M) macrophages were obtained by differentiating monocytes in the presence of IL-β (0 ng/ml) or TNFα (0 ng/ml) for 6 days. Where indicated, the LXR agonists T0907 (T09, µmol/l) and (R)-hydroxycholesterol (OH, 0 µmol/l), the PPARγ agonist GW99 (600 nmol/l) or the -LOX inhibitors cinnamyl-,- dihydroxy-a-cyanocinnamate (CDC, µg/ml) and R0089 (0 µmol/l), were added for h in medium without serum. RNA extraction and analysis Total cellular RNA was extracted from macrophages using Trizol (Life Technologies, France). RNA extraction from LCM-isolated samples was performed using the Picopure RNA extraction kit (MDS Analytical Technologies). RNA quality was assessed using the Agilent 00 Bioanalyser (Agilent Technologies). Only samples displaying a RNA Integrity Number (RIN) > 6, indicating RNA integrity in the frozen tissue, were used for RNA analysis after LCM. RNA was amplified in two rounds using the ExpressArt TRinucleotide mrna amplification Nano kit (AmpTec GmbH). For quantitative PCR (Q-PCR), RNA was reverse transcribed using random hexamer primers and Superscript reverse transcriptase (Life Technologies, France) and cdnas were quantified on a MX000 apparatus (Stratagene) using specific primers (online table ). mrna levels were normalized to cyclophilin mrna. Immunohistochemical analysis and laser capture microdissection (LCM) Immediately after endarterectomy, carotid atherosclerotic plaques were frozen in liquid nitrogen. Serial 8 µm-cryosections were stained with Oil Red O for detection of lipids. For immunohistochemical analysis, endogenous peroxidase activity was quenched and sections stained with monoclonal anti-human CD68 (Dako Corporation), goat polyclonal anti-human MR, anti-human LXRα (SantaCruz Biotechnology) or rabbit polyclonal anti-human ABCA (Novus) or anti-human -LOX (abcam) antibodies followed by detection with appropriate biotinylated secondary antibodies and streptavidin-horseradish peroxidase. Immunostains were visualized using the AEC substratechromogen system.
2 For double immunostaining, MR and -LOX were stained with N-Histofine Simple Stain MAX PO and N-Histofine Simple Stain AP, respectively (Nichirei Biosciences Inc.). MR was revealed by peroxydase and AEC which give a red/brown precipitate and -LOX by alkaline phosphatase and BCIP/NBT yielding a blue colour. Macrophage populations were defined as expressing (CD68+MR+) or not (CD68+MR-) the mannose receptor (MR) protein. Adjacent cryosections of atherosclerotic plaques containing CD68+MR+ and CD68+MR- macrophage-rich areas were dehydrated in a series of graded alcohols, cleared in xylene and prepared for laser capture microdissection (LCM), performed on an ArcturusXT Microdissection Instrument with CapSure Macro LCM Caps (MDS Analytical Technologies). Macrophage-rich areas were captured from adjacent µm-sections and pooled for RNA extraction. Cholesterol efflux Macrophages were cholesterol-loaded by incubation with [ H]cholesterol-AcLDL (0 µg/ml) for 8h. Cells were washed in PBS and cholesterol efflux measured by adding fresh medium containing or not 0 µg/ml of apoai or HDL for h. [ H]-cholesterol radioactivity was measured by scintillation counting in centrifuged medium and in cells after extraction of lipids with hexane/isopropanol (v:v). Cellular cholesterol measurement Human macrophages were and cholesterol-loaded by incubation with [ H]cholesterol-AcLDL (0 µg/ml) in RPMI 60 medium for 8 hours. For cholesterol distribution analysis, intracellular lipids were separated by thin layer chromatography (TLC) in petroleum ether/ diethyl ether/ acetic acid (80:0:0, vol:vol:vol). Spots corresponding to CE and FC were scraped and radioactivity measured by scintillation counting and the ratio CE/total radioactivity was calculated. Measurement of cholesteryl ester formation Cholesteryl ester formation within the cells was assessed by measuring the incorporation of [ C]-oleate into cholesteryl esters. Macrophages were cholesterol-loaded with AcLDL (0 µg/ml) in RPMI 60 medium for 8 hours and subsequently incubated for h with µci [ C]-oleic acid per ml of medium presented to the cells as BSA-sodium oleate complex. At the end of the assay, cells were washed and lipids extracted with hexane/isopropanol. [ C]-oleic acid incorporation into cholesteryl esters was measured on lipid extracts after separation by TLC in hexane/diethyl ether/acetic acid (90:0:, vol:vol:vol). Spots corresponding to cholesteryl oleate and oleic acid were scraped and radioactivity measured by scintillation counting. In vitro phagocytosis assay
3 Phagocytosis tests were performed using apoptotic cells or latex beads. Jurkat cells were cultured in RPMI with 0% FCS, % glutamine and % gentamicin at a concentration of x0 6 cells/ml, submitted to UV light ( nm) for min and then cultured at 7 C in % CO for h. Apoptosis was measured by flow cytometry using double staining Annexin V (AnnV) and propidium iodide (PI) to discriminate apoptotic from necrotic cells. Apoptotic cells, defined as AnnV+/PI-, were labeled with the red fluorescent dye PKH6 (Sigma). In other experiments, µm-fluorescent blue-green latex beads (Molecular Probes) were used. Apoptotic cells or latex beads were added to macrophages for h at 7 C. Where indicated, a thrombospondin (TSP-) blocking antibody (Lab Vision Corporation) was added 0min before phagocytosis assays. Macrophages (0000/condition) were then washed to remove non-phagocytosized material, scraped in PBS with % ethylene diamine tetraacetic acid (EDTA) and then analyzed on a Facscalibur flow cytometer (BD Biosciences) using Cell Quest software. Data analysis was performed with FlowJo software. Results are given as a percentage of fluorescent macrophages which have engulfed apoptotic cells or beads, reported to the total macrophage population. Each experiment was performed three times. REFERENCES. Chinetti G, Griglio S, Antonucci M, Pineda Torra I, Delerive P, Majd Z, Fruchart JC, Chapman J, Najib J, Staels B. Activation of peroxisome proliferator-activated receptors α and γ induces apoptosis of human monocyte-derived macrophages. Journal of Biological Chemistry. 998;7: Schmitz G, Niemann R, Brennhausen B, Krause R, Assmann G. Regulation of high density lipoprotein receptors in cultured macrophages: role of acyl-coa: cholesterol acyltransferase. EMBO Journal. 98;:
4 Online Table I. Primer sequences AMAC- MR IL-Ra CPT- LXRα LXRβ LOX- CD6 ApoE SR-A ABCA ACAT- LAL MMP-9 Cqa Cqb Cqc GAS-6 MERTK TSP- CD7 cyclophilin forward - AGC TCT GCT GCC TCG TCT AT- reverse -CCC ACT TCT TAT TGG GGT CA- forward '-CGA GGA AGA GGT TCG GTT CAC C-' reverse '-GCA ATC CCG GTT CTC ATG GC-' forward '-TTG AGC CTC ATG CTC TGT TC-' reverse '-CAG TGA TGT TAA CTG CCT CCA G-' forward -ACA GTC GGT GAG GCC TCT TAT GAAreverse -TCT TGC TGC CTG AAT GTG AGT TGG- forward '-AGG GCT GCA AGG GAT TCT TCC -' reverse '-TCT GAC AGC ACA CAC TCC TCC C-' forward -CTC AGT CCA GGA GAT CGT GG- reverse -CAC TCT GTC TCG TGG TTG TAG- forward -GGT CCT TTG CCT GGG ATT AGT AG- reverse -CTG GTG AGT TAG GTT TGC TTG C- forward '-TCA GCA AAT GCA AAG AAG GGA GAC-' reverse '-GGT TGA CCT GCA GCC GTT TTG-' forward -TCA GCT CCC AGG TCA CCC AG- reverse -GCG CCG GCT GCA CCT TG- forward -GAT TGG GAA CAT TCT CAG ACC TT- reverse -CTT GTC CAA AGT GAG CTG CCT T- forward -AAG GTC TTG TTC ACC TCA GCC ATG AC- reverse -GTG AAC AGC TCC AGC TCC TCC AC- forward -AGT TGA CAG CAG AGG CAG AG - reverse -GGA TAA AGA GAA TGA GGA GGG- forward '-GCA ACA GCA GAG GAA ATA C-' reverse '-GAG AAT GAC CCA CAT AAT ACA C-' forward '-GAC GAT GAC GAG TTG TGG TCC-' reverse '-CCT CGA AGA TGA AGG GGA AG-' forward '-GGA AGA ACC GTA CCA GAA CCA C-' reverse '-AGA CGA TGG ACA GGC AGA TTT C-' forward '-AGT TCG GAG AGA AGG GAG ACC-' reverse '-GAT TTT CTG GGT GGC CTT GTA G-' forward '-CAA GCC AAC ACA GGC TGC TAC-' reverse '-CTT CTG CCC TTT GGG TCC TC-' forward '-GAA AGT GAA CAC GAG GAT GCA G-' reverse '-AGC CAC GAC TTC TAC TTC CCA G-' forward '-GCT TCC TTC AGC ATA ACC AGT G-' reverse '-TTC ATG CTC TCA GGC TGC TTA G-' forward '-GCT GCA GAA TGT GAG GTT TGT C-' reverse '-GCC AAT GTA GTT AGT GCG GAT G-' forward '-GAA GGT GAA ACG ATC ATC GAG C-' reverse '-TGT CCC CAG AAC AGG AGT ATA GC-' forward '-GCA TAC GGG TCC TGG CAT CTT GTC C-' reverse '-ATG GTG ATC TTC TTG CTG GTC TTG C-'
5 A B C M DiI LDL µg cholesterol / mg protein control LDL Cellular lipid droplet surface (µm²) Online Figure I. Lower accumulation of native LDL in M macrophages Panel A. or M macrophages were incubated with DiI-native LDL (0. mg/ml) for hours and visualized using a fluorescent microscope. Panel B. or M macrophages were incubated with native LDL ( mg/ml) for hours; lipids were extracted and cellular cholesterol determined. Results are the mean ± SD of triplicate determinations, representative of independent experiments. Statistically significant differences are indicated (t-test; p< 0.0, p< 0.0, p< 0.00). Panel C. and M macrophages were loaded with native LDL ( mg/ml) for hours.oil Red O staining was performed and lipid droplet surface measured using Quips software (Leica). Median values are indicated. Statistically significant differences are indicated (t-test; p< 0.00). M LDL M M LDL
6 control LDL LDL & PMA M Online Figure II. Lower accumulation of native LDL in M macrophages is not due to a defective endocytosis and M macrophages were loaded with native LDL ( mg/ml) for hours in the absence or in the presence of PMA ( µg/ml). Oil Red O staining was performed. 6
7 MMP-9 / cyclophilin mrna A LAL / cyclophilin mrna B ACAT- / cyclophilin mrna C ILβ TNFα IL ILβ TNFα IL ILβ TNFα IL ABCA / cyclophilin mrna.. 0. D ApoE/ cyclophilin mrna E LXRα / cyclophilin mrna. 0. F ILβ TNFα IL ILβ TNFα IL ILβ TNFα IL Online Figure III. Regulation of gene expression in M and M macrophages Q-PCR analysis of MMP-9 (A), LAL (B), ACAT- (C), ABCA (D), ApoE (E) and LXRα (F) was performed on, M and M macrophages obtained by differentiation in the presence of IL-β and TNFα. mrna levels was normalized to cyclophilin mrna and expressed relative to set at. Results are means ± SD of three independent experiments. Statistically significant differences are indicated (t-test; p< 0.0, p< 0.0, p< 0.00). 7
8 MR / cyclophilin mrna T09 M M + T09 Online Figure IV. LXR activation does not affect the alternative macrophage differentiation marker MR Q-PCR analysis of MR was performed in or M macrophages in the absence or in the presence of the T0907 compound (T09, µmol/l). mrna levels was normalized to cyclophilin mrna and expressed relative to set at. Results are means ± SD of three independent experiments. Statistically significant differences are indicated (t-test; p< 0.00). 8
9 LXRα / cyclophilin mrna A B C ABCA / cyclophilin mrna ApoE / cyclophilin mrna OH M M + OH + OH M M + OH + OH M M + OH Online Figure V. M macrophage response to a natural LXR ligand Q-PCR analysis of LXRa (A), ABCA (B) and ApoE (C) was performed in or M macrophages treated or not with the -hydroxycholesterol (OH, 0 µmol/l). mrna levels was normalized to cyclophilin mrna and expressed relative to set at. Results are means ± SD of three independent experiments. Statistically significant differences are indicated (t-test; vs M p< 0.0, p< 0.0; and OH treated vs control p< 0.0, p< 0.00). 9
10 -LOX / cyclophilin mrna M Online Figure VI. -LOX gene expression is higher in alternative M macrophages Q-PCR analysis of -LOX was performed in and M macrophages. mrna levels was normalized to cyclophilin mrna and expressed relative to set at. Results are means ± SD of three independent experiments. Statistically significant differences are indicated (t-test; p< 0.00). 0
11 0 I M J M Online Figure VII. Expression of genes involved in phagocytosis is altered in alternative macrophages in vitro and in vivo. Q-PCR analysis of Cqa, Cqb, Cqc, GAS-6, MERTK and TSP- in LCM-isolated CD68+MR- and CD68+MR+ macrophage-rich areas from 7 samples (A-F) or in and M IL--differentiated macrophages (G-M). For in vitro analysis, mrna levels were normalized to cyclophilin mrna and expressed as mean ± SD relative to set at, from three independent experiments. Statistically significant differences are indicated (t-test; p< 0.00). For in vivo analysis, mrna levels were normalized to cyclophilin mrna and expressed relative to the levels in CD68+MR- area set at. Each point corresponds to a single atherosclerotic plaque. The median value is shown. Statistically significant differences are indicated (t-test; p< 0.0). G M H M M GAS-6 / cyclophilin mrna TSP- / cyclophilin mrna Cqb / cyclophilin mrna C D L CD68+MR+ CD68+MR+ M Cqc / cyclophilin mrna MERTK / cyclophilin mrna Cqa / cyclophilin mrna (fold over CD68+MR- set at ) GAS-6 / cyclophilin mrna (fold over CD68+MR- set at ) CD68+MR+ CD68+MR+ 8 6 B Cqb / cyclophilin mrna (fold over CD68+MR- set at ) A E F 0 MERTK / cyclophilin mrna Cqa / cyclophilin mrna Cqc / cyclophilin mrna (fold over CD68+MR- set at ) (fold over CD68+MR- set at ) TSP- / cyclophilin mrna (fold over CD68+MR- set at ) CD68+MR+ CD68+MR+ M
12 00 80 events FITC 0 0 without beads + GW99, beads without antibody (.%) + GW99, beads with antibody (7.%) Online Figure VIII. PPARγ-enhanced phagocytic activity of alternative macrophages is mediated by TSP-. Phagocytosis of fluorescent beads was measured in M macrophages treated with GW99 (600 nmol/l) for h in the absence or in the presence of a TSP- blocking antibody added 0 minutes before phagocytosis assay.
Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed.
Supplemental Figure 1. DLKI-DIO3 mirna/mrna complementarity. Complementarity between the indicated DLK1-DIO3 cluster mirnas and the UTR of SOX2, SOX9, HIF1A, ZEB1, ZEB2, STAT3 and CDH1with mirsvr and PhastCons
c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP
Supplementary Figure 1 Establishment of the gain- and loss-of-function experiments and cell survival assays. a Relative expression of mature mir-484 30 20 10 0 **** **** NCP mir- 484P NCP mir- 484P b Relative
a) Primary cultures derived from the pancreas of an 11-week-old Pdx1-Cre; K-MADM-p53
1 2 3 4 5 6 7 8 9 10 Supplementary Figure 1. Induction of p53 LOH by MADM. a) Primary cultures derived from the pancreas of an 11-week-old Pdx1-Cre; K-MADM-p53 mouse revealed increased p53 KO/KO (green,
Supplementary Materials
Supplementary Materials 1 Supplementary Table 1. List of primers used for quantitative PCR analysis. Gene name Gene symbol Accession IDs Sequence range Product Primer sequences size (bp) β-actin Actb gi
Supplemental Data. Shin et al. Plant Cell. (2012) /tpc YFP N
MYC YFP N PIF5 YFP C N-TIC TIC Supplemental Data. Shin et al. Plant Cell. ()..5/tpc..95 Supplemental Figure. TIC interacts with MYC in the nucleus. Bimolecular fluorescence complementation assay using
Abbreviations: P- paraffin-embedded section; C, cryosection; Bio-SA, biotin-streptavidin-conjugated fluorescein amplification.
Supplementary Table 1. Sequence of primers for real time PCR. Gene Forward primer Reverse primer S25 5 -GTG GTC CAC ACT ACT CTC TGA GTT TC-3 5 - GAC TTT CCG GCA TCC TTC TTC-3 Mafa cds 5 -CTT CAG CAA GGA
CD31 5'-AGA GAC GGT CTT GTC GCA GT-3' 5 ' -TAC TGG GCT TCG AGA GCA GT-3'
Table S1. The primer sets used for real-time RT-PCR analysis. Gene Forward Reverse VEGF PDGFB TGF-β MCP-1 5'-GTT GCA GCA TGA ATC TGA GG-3' 5'-GGA GAC TCT TCG AGG AGC ACT T-3' 5'-GAA TCA GGC ATC GAG AGA
Supplementary Appendix
Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Sherman SI, Wirth LJ, Droz J-P, et al. Motesanib diphosphate
Supplementary Figure 1 a
Supplementary Figure a Normalized expression/tbp (A.U.).6... Trip-br transcripts Trans Trans Trans b..5. Trip-br Ctrl LPS Normalized expression/tbp (A.U.) c Trip-br transcripts. adipocytes.... Trans Trans
Supplementary Document
Supplementary Document 1. Supplementary Table legends 2. Supplementary Figure legends 3. Supplementary Tables 4. Supplementary Figures 5. Supplementary References 1. Supplementary Table legends Suppl.
Toluidin-Staining of mast cells Ear tissue was fixed with Carnoy (60% ethanol, 30% chloroform, 10% acetic acid) overnight at 4 C, afterwards
Toluidin-Staining of mast cells Ear tissue was fixed with Carnoy (60% ethanol, 30% chloroform, 10% acetic acid) overnight at 4 C, afterwards incubated in 100 % ethanol overnight at 4 C and embedded in
Supplementary Figure 1. ROS induces rapid Sod1 nuclear localization in a dosagedependent manner. WT yeast cells (SZy1051) were treated with 4NQO at
Supplementary Figure 1. ROS induces rapid Sod1 nuclear localization in a dosagedependent manner. WT yeast cells (SZy1051) were treated with 4NQO at different concentrations for 30 min and analyzed for
Figure S1. Analysis of genomic and cdna sequences of the targeted regions in WT-KI and
Figure S1. Analysis of genomic and sequences of the targeted regions in and indicated mutant KI cells, with WT and corresponding mutant sequences underlined. (A) cells; (B) K21E-KI cells; (C) D33A-KI cells;
Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1
Supplementary Figure 1 U1 inhibition causes a shift of RNA-seq reads from exons to introns. (a) Evidence for the high purity of 4-shU-labeled RNAs used for RNA-seq. HeLa cells transfected with control
Supplementary Figures
Supplementary Figures Supplementary Figure 1. H3F3B expression in lung cancer. a. Comparison of H3F3B expression in relapsed and non-relapsed lung cancer patients. b. Prognosis of two groups of lung cancer
Supplementary Figure 1 MicroRNA expression in human synovial fibroblasts from different locations. MicroRNA, which were identified by RNAseq as most
Supplementary Figure 1 MicroRNA expression in human synovial fibroblasts from different locations. MicroRNA, which were identified by RNAseq as most differentially expressed between human synovial fibroblasts
Citation for published version (APA): Oosterveer, M. H. (2009). Control of metabolic flux by nutrient sensors Groningen: s.n.
University of Groningen Control of metabolic flux by nutrient sensors Oosterveer, Maaike IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it.
Astaxanthin prevents and reverses diet-induced insulin resistance and. steatohepatitis in mice: A comparison with vitamin E
Supplementary Information Astaxanthin prevents and reverses diet-induced insulin resistance and steatohepatitis in mice: A comparison with vitamin E Yinhua Ni, 1,2 Mayumi Nagashimada, 1 Fen Zhuge, 1 Lili
Table S1. Oligonucleotides used for the in-house RT-PCR assays targeting the M, H7 or N9. Assay (s) Target Name Sequence (5 3 ) Comments
SUPPLEMENTAL INFORMATION 2 3 Table S. Oligonucleotides used for the in-house RT-PCR assays targeting the M, H7 or N9 genes. Assay (s) Target Name Sequence (5 3 ) Comments CDC M InfA Forward (NS), CDC M
Supplementary Table 2. Conserved regulatory elements in the promoters of CD36.
Supplementary Table 1. RT-qPCR primers for CD3, PPARg and CEBP. Assay Forward Primer Reverse Primer 1A CAT TTG TGG CCT TGT GCT CTT TGA TGA GTC ACA GAA AGA ATC AAT TC 1B AGG AAA TGA ACT GAT GAG TCA CAG
Plasmids Western blot analysis and immunostaining Flow Cytometry Cell surface biotinylation RNA isolation and cdna synthesis
Plasmids psuper-retro-s100a10 shrna1 was constructed by cloning the dsdna oligo 5 -GAT CCC CGT GGG CTT CCA GAG CTT CTT TCA AGA GAA GAA GCT CTG GAA GCC CAC TTT TTA-3 and 5 -AGC TTA AAA AGT GGG CTT CCA GAG
Nature Immunology: doi: /ni.3836
Supplementary Figure 1 Recombinant LIGHT-VTP induces pericyte contractility and endothelial cell activation. (a) Western blot showing purification steps for full length murine LIGHT-VTP (CGKRK) protein:
Cross-talk between mineralocorticoid and angiotensin II signaling for cardiac
ONLINE SUPPLEMENT TO Crosstalk between mineralocorticoid and angiotensin II signaling for cardiac remodeling An Di ZHANG,,3, Aurelie NGUYEN DINH CAT*,,3, Christelle SOUKASEUM *,,3, Brigitte ESCOUBET, 4,
A smart acid nanosystem for ultrasensitive. live cell mrna imaging by the target-triggered intracellular self-assembly
Electronic Supplementary Material (ESI) for Chemical Science. This journal is The Royal Society of Chemistry 2017 A smart ZnO@polydopamine-nucleic acid nanosystem for ultrasensitive live cell mrna imaging
BIOLOGY 621 Identification of the Snorks
Name: Date: Block: BIOLOGY 621 Identification of the Snorks INTRODUCTION: In this simulation activity, you will examine the DNA sequence of a fictitious organism - the Snork. Snorks were discovered on
BHP 2-7 and Nthy-ori 3-1 cells were grown in RPMI1640 medium (Hyclone) supplemented with 10% fetal bovine serum (Gibco), 2mM L-glutamine, and 100 U/mL
1 2 3 4 Materials and Methods Cell culture BHP 2-7 and Nthy-ori 3-1 cells were grown in RPMI1640 medium (Hyclone) 5 supplemented with 10% fetal bovine serum (Gibco), 2mM L-glutamine, and 100 U/mL 6 penicillin-streptomycin.
ice-cold 70% ethanol with gentle vortexing, incubated at -20 C for 4 hours, and washed with PBS.
Cell cycle analysis For cell cycle analysis, single cell suspensions of E12.5 fetal liver cells were suspended in 4 ml ice-cold 7% ethanol with gentle vortexing, incubated at -2 C for 4 hours, and washed
SUPPLEMENTARY DATA. Supplementary Table 1. Primer sequences for qrt-pcr
Supplementary Table 1. Primer sequences for qrt-pcr Gene PRDM16 UCP1 PGC1α Dio2 Elovl3 Cidea Cox8b PPARγ AP2 mttfam CyCs Nampt NRF1 16s-rRNA Hexokinase 2, intron 9 β-actin Primer Sequences 5'-CCA CCA GCG
Supplementary Figure 1
Supplementary Figure 1 Supplementary Figure 1. Lats1/2 deleted ihbs and ihps showed decreased transcripts of hepatocyte related genes (a and b) Western blots (a) and recombination PCR (b) of control and
SUPPLEMENTARY INFORMATION
doi: 10.1038/nature05883 SUPPLEMENTARY INFORMATION Supplemental Figure 1 Prostaglandin agonists and antagonists alter runx1/cmyb expression. a-e, Embryos were exposed to (b) PGE2 and (c) PGI2 (20μM) and
Supplementary Figure 1
Metastatic melanoma Primary melanoma Healthy human skin Supplementary Figure 1 CD22 IgG4 Supplementary Figure 1: Immunohisochemical analysis of CD22+ (left) and IgG4 (right), cells (shown in red and indicated
McAlpine PERK-GSK3 regulates foam cell formation. Supplemental Material. Supplementary Table I. Sequences of real time PCR primers.
Mclpine PERK-GSK3 regulates foam cell formation Supplemental Material Supplementary Table I. Sequences of real time PCR primers. Primer Name Primer Sequences (5-3 ) Product Size (bp) GRP78 (human) Fwd:
Supplementary Figure 1a
Supplementary Figure 1a Hours: E-cadherin TGF-β On TGF-β Off 0 12 24 36 48 24 48 72 Vimentin βactin Fig. S1a. Treatment of AML12 cells with TGF-β induces EMT. Treatment of AML12 cells with TGF-β results
Journal of Cell Science Supplementary information. Arl8b +/- Arl8b -/- Inset B. electron density. genotype
J. Cell Sci. : doi:.4/jcs.59: Supplementary information E9. A Arl8b /- Arl8b -/- Arl8b Arl8b non-specific band Gapdh Tbp E7.5 HE Inset B D Control al am hf C E Arl8b -/- al am hf E8.5 F low middle high
Culture Density (OD600) 0.1. Culture Density (OD600) Culture Density (OD600) Culture Density (OD600) Culture Density (OD600)
A. B. C. D. E. PA JSRI JSRI 2 PA DSAM DSAM 2 DSAM 3 PA LNAP LNAP 2 LNAP 3 PAO Fcor Fcor 2 Fcor 3 PAO Wtho Wtho 2 Wtho 3 Wtho 4 DTSB Low Iron 2 4 6 8 2 4 6 8 2 22 DTSB Low Iron 2 4 6 8 2 4 6 8 2 22 DTSB
Phylogenetic analysis of human and chicken importins. Only five of six importins were studied because
Supplementary Figure S1 Phylogenetic analysis of human and chicken importins. Only five of six importins were studied because importin-α6 was shown to be testis-specific. Human and chicken importin protein
Supplementary Materials and Methods
DD2 suppresses tumorigenicity of ovarian cancer cells by limiting cancer stem cell population Chunhua Han et al. Supplementary Materials and Methods Analysis of publicly available datasets: To analyze
Supplementary Figure 1
Supplementary Figure 1 Supplementary Figure 1: Cryopreservation alters CD62L expression by CD4 T cells. Freshly isolated (left) or cryopreserved PBMCs (right) were stained with the mix of antibodies described
Supplementary information
Supplementary information Unique polypharmacology nuclear receptor modulator blocks inflammatory signaling pathways Mi Ra Chang 1, Anthony Ciesla 1, Timothy S. Strutzenberg 1, Scott J. Novick 1, Yuanjun
INTERLEUKIN-10 FACILITATES BOTH CHOLESTEROL UPTAKE AND EFFLUX IN MACROPHAGES
SUPPLEMENTAL DATA INTERLEUKIN-10 FACILITATES BOTH CHOLESTEROL UPTAKE AND EFFLUX IN MACROPHAGES Xinbing Han, Shiro Kitamoto, Qingyu Lian and William A. Boisvert Vascular Medicine Research Unit, Brigham
Supplemental Information. Cancer-Associated Fibroblasts Neutralize. the Anti-tumor Effect of CSF1 Receptor Blockade
Cancer Cell, Volume 32 Supplemental Information Cancer-Associated Fibroblasts Neutralize the Anti-tumor Effect of CSF1 Receptor Blockade by Inducing PMN-MDSC Infiltration of Tumors Vinit Kumar, Laxminarasimha
Supplementary Figure 1
Supplementary Figure 1 3 3 3 1 1 Bregma -1.6mm 3 : Bregma Ref) Http://www.mbl.org/atlas165/atlas165_start.html Bregma -.18mm Supplementary Figure 1 Schematic representation of the utilized brain slice
Supporting Information
Supporting Information Malapeira et al. 10.1073/pnas.1217022110 SI Materials and Methods Plant Material and Growth Conditions. A. thaliana seedlings were stratified at 4 C in the dark for 3 d on Murashige
www.lessonplansinc.com Topic: Protein Synthesis - Sentence Activity Summary: Students will simulate transcription and translation by building a sentence/polypeptide from words/amino acids. Goals & Objectives:
Supplemental Information. Th17 Lymphocytes Induce Neuronal. Cell Death in a Human ipsc-based. Model of Parkinson's Disease
Cell Stem Cell, Volume 23 Supplemental Information Th17 Lymphocytes Induce Neuronal Cell Death in a Human ipsc-based Model of Parkinson's Disease Annika Sommer, Franz Maxreiter, Florian Krach, Tanja Fadler,
CIRCRESAHA/2004/098145/R1 - ONLINE 1. Validation by Semi-quantitative Real-Time Reverse Transcription PCR
CIRCRESAHA/2004/098145/R1 - ONLINE 1 Expanded Materials and Methods Validation by Semi-quantitative Real-Time Reverse Transcription PCR Expression patterns of 13 genes (Online Table 2), selected with respect
SUPPORTING INFORMATION
SUPPORTING INFORMATION Biology is different in small volumes: endogenous signals shape phenotype of primary hepatocytes cultured in microfluidic channels Amranul Haque, Pantea Gheibi, Yandong Gao, Elena
PATIENTS AND METHODS. Subjects
PATIENTS AND METHODS Subjects Twenty-nine morbidly obese subjects involved in a gastric surgery program were enrolled in the study between October 25 and March 21. Bariatric surgery was performed in patients
Description of Supplementary Files. File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables
Description of Supplementary Files File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables Supplementary Figure 1: (A), HCT116 IDH1-WT and IDH1-R132H cells were
A basic helix loop helix transcription factor controls cell growth
A basic helix loop helix transcription factor controls cell growth and size in root hairs Keke Yi 1,2, Benoît Menand 1,3, Elizabeth Bell 1, Liam Dolan 1,4 Supplementary note Low soil phosphate availability
Supplementary Information. Bamboo shoot fiber prevents obesity in mice by. modulating the gut microbiota
Supplementary Information Bamboo shoot fiber prevents obesity in mice by modulating the gut microbiota Xiufen Li 1,2, Juan Guo 1, Kailong Ji 1,2, and Ping Zhang 1,* 1 Key Laboratory of Tropical Plant Resources
Loyer, et al. microrna-21 contributes to NASH Suppl 1/15
Loyer, et al. microrna-21 contributes to NASH Suppl 1/15 SUPPLEMENTARY MATERIAL: Liver MicroRNA-21 is Overexpressed in Non Alcoholic Steatohepatitis and Contributes to the Disease in Experimental Models
Formylpeptide receptor2 contributes to colon epithelial homeostasis, inflammation, and tumorigenesis
Supplementary Data Formylpeptide receptor2 contributes to colon epithelial homeostasis, inflammation, and tumorigenesis Keqiang Chen, Mingyong Liu, Ying Liu, Teizo Yoshimura, Wei Shen, Yingying Le, Scott
Relationship of the APOA5/A4/C3/A1 gene cluster and APOB gene polymorphisms with dyslipidemia
elationship of the APOA5/A4/C3/A1 gene cluster and APOB gene polymorphisms with dyslipidemia H.J. Ou 1, G. Huang 2, W. Liu 3, X.L. Ma 2, Y. Wei 4, T. Zhou 5 and Z.M. Pan 3 1 Department of Neurology, The
HCV Persistence and Immune Evasion in the Absence of Memory T Cell Help.
SOM Text HCV Persistence and Immune Evasion in the Absence of Memory T Cell Help. Arash Grakoui 1, Naglaa H. Shoukry 2, David J. Woollard 2, Jin-Hwan Han 1, Holly L. Hanson 1, John Ghrayeb 3, Krishna K.
Lezione 10. Sommario. Bioinformatica. Lezione 10: Sintesi proteica Synthesis of proteins Central dogma: DNA makes RNA makes proteins Genetic code
Lezione 10 Bioinformatica Mauro Ceccanti e Alberto Paoluzzi Lezione 10: Sintesi proteica Synthesis of proteins Dip. Informatica e Automazione Università Roma Tre Dip. Medicina Clinica Università La Sapienza
Nucleotide Sequence of the Australian Bluetongue Virus Serotype 1 RNA Segment 10
J. gen. Virol. (1988), 69, 945-949. Printed in Great Britain 945 Key words: BTV/genome segment lo/nucleotide sequence Nucleotide Sequence of the Australian Bluetongue Virus Serotype 1 RNA Segment 10 By
TetR repressor-based bioreporters for the detection of doxycycline using Escherichia
Supplementary materials TetR repressor-based bioreporters for the detection of doxycycline using Escherichia coli and Acinetobacter oleivorans Hyerim Hong and Woojun Park * Department of Environmental
Supporting Information
Supporting Information Molecular Recognition Based DNA Nanoassemblies on the Surfaces of Nanosized Exosomes Shuo Wan,, Liqin Zhang,,, Sai Wang, Yuan Liu,, Cuichen Wu, Cheng Cui, Hao Sun, Muling Shi, Ying
Baseline clinical characteristics for the 81 CMML patients Routine diagnostic testing and statistical analyses... 3
Next-Generation Sequencing Technology Reveals a Characteristic Pattern of Molecular Mutations in 72.8% of Chronic Myelomonocytic Leukemia (CMML) by Detecting Frequent Alterations in TET2, CBL, RAS, and
Supplemental Figures: Supplemental Figure 1
Supplemental Figures: Supplemental Figure 1 Suppl. Figure 1. BM-DC infection with H. pylori does not induce cytotoxicity and treatment of BM-DCs with H. pylori sonicate, but not heat-inactivated bacteria,
Resistance to Tetracycline Antibiotics by Wangrong Yang, Ian F. Moore, Kalinka P. Koteva, Donald W. Hughes, David C. Bareich and Gerard D. Wright.
Supplementary Data for TetX is a Flavin-Dependent Monooxygenase Conferring Resistance to Tetracycline Antibiotics by Wangrong Yang, Ian F. Moore, Kalinka P. Koteva, Donald W. Hughes, David C. Bareich and
Development of RT-qPCR-based molecular diagnostic assays for therapeutic target selection of breast cancer patients
Development of RT-qPCR-based molecular diagnostic assays for therapeutic target selection of breast cancer patients Sangjung Park The Graduate School Yonsei University Department of Biomedical Laboratory
Supporting Information. Mutational analysis of a phenazine biosynthetic gene cluster in
Supporting Information for Mutational analysis of a phenazine biosynthetic gene cluster in Streptomyces anulatus 9663 Orwah Saleh 1, Katrin Flinspach 1, Lucia Westrich 1, Andreas Kulik 2, Bertolt Gust
Mechanistic and functional insights into fatty acid activation in Mycobacterium tuberculosis SUPPLEMENTARY INFORMATION
Mechanistic and functional insights into fatty acid activation in Mycobacterium tuberculosis Pooja Arora 1, Aneesh Goyal 2, Vivek T atarajan 1, Eerappa Rajakumara 2, Priyanka Verma 1, Radhika Gupta 3,
Single-Molecule Analysis of Gene Expression Using Two-Color RNA- Labeling in Live Yeast
Supplemental Figures, Tables and Results Single-Molecule Analysis of Gene Expression Using Two-Color RNA- Labeling in Live Yeast Sami Hocine 1, Pascal Raymond 2, Daniel Zenklusen 2, Jeffrey A. Chao 1 &
Supplementary Information
Supplementary Information Remodeling of heterochromatin structure slows neuropathological progression and prolongs survival in an animal model of Huntington s disease Junghee Lee, Yu Jin Hwang, Yunha Kim,
Beta Thalassemia Case Study Introduction to Bioinformatics
Beta Thalassemia Case Study Sami Khuri Department of Computer Science San José State University San José, California, USA sami.khuri@sjsu.edu www.cs.sjsu.edu/faculty/khuri Outline v Hemoglobin v Alpha
Beta Thalassemia Sami Khuri Department of Computer Science San José State University Spring 2015
Bioinformatics in Medical Product Development SMPD 287 Three Beta Thalassemia Sami Khuri Department of Computer Science San José State University Hemoglobin Outline Anatomy of a gene Hemoglobinopathies
*To whom correspondence should be addressed. This PDF file includes:
www.sciencemag.org/cgi/content/full/science.1212182/dc1 Supporting Online Material for Partial Retraction to Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients with Chronic Fatigue
SUPPLEMENTARY INFORMATION
BASELINE ISCHAEMIA a b Phd2 +/- c d Collateral growth and maintenance SMC recruitment SMC proliferation Phd2 +/- NF- B off NF- B on NF- B on NF- B on Endothelial cell Smooth muscle cell Pro-arteriogenic
SUPPLEMENTARY RESULTS
SUPPLEMENTARY RESULTS Supplementary Table 1. hfpr1- Flpln-CHO hfpr2-flpln-cho pec 50 E max (%) Log( /K A) Log( /K A) N pec 50 E max (%) Log( /K A) Log( /K A) n ERK1/2 phosphorylation fmlp 9.0±0.6 80±7
Isolate Sexual Idiomorph Species
SUPLEMENTARY TABLE 1. Isolate identification, sexual idiomorph and species of each isolate used for MAT locus distribution in Paracoccidioides species. Isolate Sexual Idiomorph Species Pb01 MAT1-1 P. lutzii
Enhanced detection and serotyping of Streptococcus pneumoniae using multiplex polymerase chain reaction
Original article http://dx.doi.org/10.3345/kjp.2012.55.11.424 Korean J Pediatr 2012;55(11):424-429 eissn 1738-1061 pissn 2092-7258 Enhanced detection and serotyping of Streptococcus pneumoniae using multiplex
Mutation Screening and Association Studies of the Human UCP 3 Gene in Normoglycemic and NIDDM Morbidly Obese Patients
Mutation Screening and Association Studies of the Human UCP 3 Gene in Normoglycemic and NIDDM Morbidly Obese Patients Shuichi OTABE, Karine CLEMENT, Séverine DUBOIS, Frederic LEPRETRE, Veronique PELLOUX,
Mutation analysis of a Chinese family with oculocutaneous albinism
/, 2016, Vol. 7, (No. 51), pp: 84981-84988 Mutation analysis of a Chinese family with oculocutaneous albinism Xiong Wang 1, Yaowu Zhu 1, Na Shen 1, Jing Peng 1, Chunyu Wang 1, Haiyi Liu 2, Yanjun Lu 1
Expression of Selected Inflammatory Cytokine Genes in Bladder Biopsies
Borneo Journal of Resource Science and Technology (2013) 3(2): 15-20 Expression of Selected Inflammatory Cytokine Genes in Bladder Biopsies EDMUND UI-HANG SIM *1, NUR DIANA ANUAR 2, TENG-AIK ONG 3, GUAN-
Supplementary Materials for
www.sciencesignaling.org/cgi/content/full/10/473/eaai7696/dc1 Supplementary Materials for Astrocyte-shed extracellular vesicles regulate the peripheral leukocyte response to inflammatory brain lesions
Advanced Subsidiary Unit 1: Lifestyle, Transport, Genes and Health
Write your name here Surname Other names Edexcel GCE Centre Number Candidate Number Biology Advanced Subsidiary Unit 1: Lifestyle, Transport, Genes and Health Thursday 8 January 2009 Morning Time: 1 hour
The Clinical Performance of Primary HPV Screening, Primary HPV Screening Plus Cytology Cotesting, and Cytology Alone at a Tertiary Care Hospital
The Clinical Performance of Primary HPV Screening, Primary HPV Screening Plus Cytology Cotesting, and Cytology Alone at a Tertiary Care Hospital Jung-Woo Choi MD, PhD; Younghye Kim MD, PhD; Ju-Han Lee
University of Groningen. Vasoregression in incipient diabetic retinopathy Pfister, Frederick
University of Groningen Vasoregression in incipient diabetic retinopathy Pfister, Frederick IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
Cancer Genetics 204 (2011) 45e52
Cancer Genetics 204 (2011) 45e52 Exon scanning by reverse transcriptaseepolymerase chain reaction for detection of known and novel EML4eALK fusion variants in nonesmall cell lung cancer Heather R. Sanders
SUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION Autonomous Multistep Organic Synthesis in a Single Isothermal Solution Mediated by a DNA Walker Yu He and David R. Liu* Supplementary Methods General Methods. DNA oligonucleotides
University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA
Primary research Cytokine, activation marker, and chemokine receptor expression by individual CD4 + memory T cells in rheumatoid arthritis synovium Toshihiro Nanki and Peter E Lipsky University of Texas
Viral hepatitis, which affects half a billion people
GASTROENTEROLOGY 2006;130:435 452 BASIC LIVER, PANCREAS, AND BILIARY TRACT Natural Killer Cells Ameliorate Liver Fibrosis by Killing Activated Stellate Cells in NKG2D-Dependent and Tumor Necrosis Factor
Detection of 549 new HLA alleles in potential stem cell donors from the United States, Poland and Germany
HLA ISSN 2059-2302 BRIEF COMMUNICATION Detection of 549 new HLA alleles in potential stem cell donors from the United States, Poland and Germany C. J. Hernández-Frederick 1, N. Cereb 2,A.S.Giani 1, J.
SUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION doi:10.1038/nature10743 Supplementary Figures and Legends Supplementary Figure 1. CYP17A1 (red boxes) lies at the intersection of steroid hormone biosynthetic pathways. CYP17A1
Supplementary information
Supplementary information Full methods The conduct of the study was approved by an NHS research ethical committee prior to commencement (reference 12/WS/0288) and was conducted according to the principles
Supplementary Figure S1
Supplementry Figure S Tissue weights (g).... Liver Hert Brin Pncres Len mss (g) 8 6 -% +% 8 6 Len mss Len mss (g) (% ody weight) Len mss (% ody weight) c Tiilis nterior weight (g).6...... Qudriceps weight
Isolation and Genetic Characterization of New Reassortant H3N1 Swine Influenza Virus from Pigs in the Midwestern United States
JOURNAL OF VIROLOGY, May 2006, p. 5092 5096 Vol. 80, No. 10 0022-538X/06/$08.00 0 doi:10.1128/jvi.80.10.5092 5096.2006 Copyright 2006, American Society for Microbiology. All Rights Reserved. Isolation
3) Table_S1: Clinical Characteristics of Breast Cancer Patients. 5) Table_S3: Primer sequences used for qt-pcr of ChIP samples
Supplemental Section: 1) Eight supplemental figures and legends 2) Supplemental Materials and Methods 3) Table_S1: Clinical Characteristics of Breast Cancer Patients 4) Table_S2: Oligonucleotide sequences
Supplementary Material Hofko M et al., Detection of carbapenemases by real-time PCR and melt-curve analysis on the BD MAX TM System
Supplementary Material Hofko M et al., Detection of carbapenemases by real-time PCR and melt-curve analysis on the BD MAX TM System Supplementary Material and Methods Characterization of isolates by the
Supplementary Figure 1: GPCR profiling and G q signaling in murine brown adipocytes (BA). a, Number of GPCRs with 2-fold lower expression in mature
Supplementary Figure 1: GPCR profiling and G q signaling in murine brown adipocytes (BA). a, Number of GPCRs with 2-fold lower expression in mature BA vs. preadipocytes. b, Number of GPCRs with 2-fold
The synergistic effect of homocysteine and lipopolysaccharide on the differentiation and conversion of raw264.7 macrophages
Gao et al. Journal of Inflammation 2014, 11:13 RESEARCH Open Access The synergistic effect of homocysteine and lipopolysaccharide on the differentiation and conversion of raw264.7 macrophages Shanshan
Nomenclature What is in a name? My name Joseˊ Jimenez = Bill Dana John L.C. Savony = Frank Fontaine
Nomenclature What is in a name? My name Joseˊ Jimenez = Bill Dana John L.C. Savony = Frank Fontaine Three categories of Amyloid Terms 1. Amyloidologists, Amyloid Centers, Researchers official nomenclature
Nucleotide diversity of the TNF gene region in an African village
(2001) 2, 343 348 2001 Nature Publishing Group All rights reserved 1466-4879/01 $15.00 www.nature.com/gene Nucleotide diversity of the TNF gene region in an African village A Richardson 1, F Sisay-Joof
The Effect of Epstein-Barr Virus Latent Membrane Protein 2 Expression on the Kinetics of Early B Cell Infection
The Effect of Epstein-Barr Virus Latent Membrane Protein 2 Expression on the Kinetics of Early B Cell Infection Laura R. Wasil, Monica J. Tomaszewski, Aki Hoji, David T. Rowe* Department of Infectious
Figure 1. Effects of FGF21 on adipose tissue. (A) Representative histological. findings of epididymal adipose tissue (B) mrna expression of
SUPPLEMENTAL MATERIAL EN-12-2276 Figure 1. Effects of FGF21 on adipose tissue. (A) Representative histological findings of epididymal adipose tissue (B) mrna expression of adipocytokines in adipose tissue.
Ho Young Jung Hye Seung Han Hyo Bin Kim 1 Seo Young Oh 1 Sun-Joo Lee 2 Wook Youn Kim
Journal of Pathology and Translational Medicine 2016; 50: 138-146 ORIGINAL ARTICLE Comparison of Analytical and Clinical Performance of HPV 9G DNA Chip, PANArray HPV Genotyping Chip, and Hybrid-Capture
SUPPLEMENTAL FIGURE 1
SUPPLEMENTL FIGURE 1 C Supplemental Figure 1. pproach for removal of snorns from Rpl13a gene. () Wild type Rpl13a exonintron structure is shown, with exo in black and intronic snorns in red rectangles.