Journal: Clinical and Experimental Immunology

Article Title: Investigating the role of proinflammatory CD16 + monocytes in the pathogenesis of inflammatory bowel disease

doi: 10.1111/j.1365-2249.2010.04177.x

Figure Lengend Snippet: The peripheral blood CD14+ CD16+ monocyte population is increased in active Crohn's disease. (a) Peripheral blood mononuclear cells (PBMC) were stained for CD14, CD16 and CD36 and analysed by flow cytometry. Cells were pre-gated for high forward-scatter (FSC), side-scatter (SSC) and CD36 expression, and monocytes separated by CD14/CD16 expression. The lower panels show representative dot plots from a healthy volunteer, and Crohn's disease patients with quiescent [Crohn's disease activity index (CDAI)<150] and active (CDAI>150) inflammation. (b) The ratio of CD14+ CD16+ monocytes was increased significantly in Crohn's disease patients compared to healthy controls. *P = 0·01 (two-tailed Student's t-test with Welch's correction). (c) Further categorization of patients revealed that the CD14+ CD16+ population was increased in active Crohn's disease compared to healthy controls, but not quiescent disease or patients with ulcerative colitis. *P < 0·05 (Bonferroni's post-test following one-way analysis of variance).

Article Snippet: Slides were then incubated at 4°C overnight with the following primary antibodies: mouse anti-CD14 (BioLegend, San Diego, CA, USA), rat anti-CD16 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-CD36 (Novus Biologicals, Littleton, CO, USA).

Techniques: Staining, Flow Cytometry, Expressing, Activity Assay, Two Tailed Test

Journal: Clinical and Experimental Immunology

Article Title: Investigating the role of proinflammatory CD16 + monocytes in the pathogenesis of inflammatory bowel disease

doi: 10.1111/j.1365-2249.2010.04177.x

Figure Lengend Snippet: The number of CD14+ CD16+ monocytes is enhanced significantly in the lamina propria of Crohn's disease patients. (a) Monocytic lineage of CD14+ CD16+ cells in the colonic mucosa was confirmed by three-channel immunofluorescence microscopy for CD14 (green), CD16 (red) and CD36 (blue). No or limited CD36 staining was observed in CD14+ CD16– cells (i) and CD14– CD16+ cells (ii). In contrast, CD14+ CD16+ cells stained strongly for CD36 (iii, white pseudocolour). Colonic crypts are indicated with dashed lines. Lower panels, magnification of the boxed cells shown above. (b) Representative images of colonic mucosal sections from non-inflammatory bowel disease (IBD) control patients, non-inflamed and inflamed tissue from Crohn's disease patients, and non-inflamed ulcerative colitis samples. CD14+ CD16+ monocytes are shown in yellow pseudocolour, with nuclei in blue. (c) The number of CD14+ CD16+ cells per high-power field (HPF) was significantly increased in Crohn's disease mucosa, and enhanced further in actively inflamed tissue. Each data point represents the average of five or more HPF per patient sample. *P < 0·001 versus control, †P < 0·001 versus IBD – not inflamed [Bonferroni's post-test following one-way analysis of variance (anova)]. (d) The ratio of CD14+ CD16+ cells was increased significantly in Crohn's disease patients compared to controls. *P < 0·001 (Bonferroni's post-test following one-way anova). (e) The increase in total CD14+ CD16+ monocytes was confirmed by flow cytometry. Mononuclear cells were isolated from control and Crohn's disease colonic mucosa as described in the Materials and methods, pre-gated for forward-scatter (high), side-scatter (high) CD36+, and separated by CD14/CD16 expression. A dramatic increase of CD14+ CD16+ monocytes was seen in IBD tissue (red box). Plots are representative of three patient samples per group. (f) The tumour necrosis factor (TNF)-α mean fluorescence intensity (MFI, arbitrary units) of CD14+ CD16+ monocytes was significantly higher compared to CD14+ CD16– cells in the same field of vision. The graph shows the result from 14 HPF taken from three Crohn's disease patient samples. *P < 0·001 (two-tailed Student's t-test).

Article Snippet: Slides were then incubated at 4°C overnight with the following primary antibodies: mouse anti-CD14 (BioLegend, San Diego, CA, USA), rat anti-CD16 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-CD36 (Novus Biologicals, Littleton, CO, USA).

Techniques: Immunofluorescence, Microscopy, Staining, Flow Cytometry, Isolation, Expressing, Fluorescence, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Luminal Lipid Regulates CD36 Levels and Downstream Signaling to Stimulate Chylomicron Synthesis *

doi: 10.1074/jbc.M111.233551

Figure Lengend Snippet: CD36 deficiency does not alter jejunal uptake and esterification of micellar fatty acids in the mouse. Linoleic acid (0.5 ml, 2180 μm) in solution containing [1-14C]linoleic acid (51 mCi/mmol) emulsified with 10 mm taurocholic acid was infused into in situ isolated jejunal loops of fasted mice. L and M correspond to the percentage of [1-14C]linoleic acid remaining respectively in the lumen (L) and the mucosa (M). The percentage of [1-14C]linoleic disappeared (D) (mainly absorbed or oxidized) was determined by subtraction of the radioactivity remaining into M and L from the radioactivity initially infused into the lumen. The values shown are the means ± S.E. (n = 3). B, relative distribution of [1-14C] radioactivity in various lipid classes in the mucosa. PC, phosphatidylcholine; PS/PI, phosphatidylserine/phosphatidylinositol; DG/MG, di- and monoglyceride; FFA, free fatty acid.

Article Snippet: Anti-mouse CD36 (R & D System or Santa Cruz Biotechnology), mouse anti-ubiquitin (Zymed Laboratories Inc., Invitrogen), anti-phospho-ERK1/2 (Thr-202/Tyr-204) and anti-ERK1/2 (Cell Signaling Technology), anti-apoB, anti-HSC70 (Santa Cruz Biotechnology), anti-MTP (BD Sciences), secondary antibody peroxidase conjugate goat anti-rabbit (Chemicon International), anti-mouse IgG (Sigma-Aldrich), and donkey anti-goat (Santa Cruz Biotechnology) were from commercial sources.

Techniques: In Situ, Isolation, Radioactivity

Journal: The Journal of Biological Chemistry

Article Title: Luminal Lipid Regulates CD36 Levels and Downstream Signaling to Stimulate Chylomicron Synthesis *

doi: 10.1074/jbc.M111.233551

Figure Lengend Snippet: Lipid-dependent disappearance of CD36 protein from the luminal side of mice and rat jejunum. A, immunolocalization of CD36 in jejunal epithelium in mice. Fasted controls were compared with experimental mice 4 h after force-feeding with 0.5 ml oil. Immunostaining of CD36 on CD36−/− mice demonstrates specificity of the antibody (R & D System). B, immunolocalization of CD36 in jejunal epithelium in the rat. Fasted controls were compared with rats refed for 6 h a standard laboratory chow containing 3% lipids (w/w) or a lipid-free diet. Specific CD36 immunostaining was performed using an antiserum raised in rabbit against purified rat CD36 and a FITC-conjugated anti-rabbit antibody raised in goat. In negative control micrographs, the primary antibody was omitted.

Article Snippet: Anti-mouse CD36 (R & D System or Santa Cruz Biotechnology), mouse anti-ubiquitin (Zymed Laboratories Inc., Invitrogen), anti-phospho-ERK1/2 (Thr-202/Tyr-204) and anti-ERK1/2 (Cell Signaling Technology), anti-apoB, anti-HSC70 (Santa Cruz Biotechnology), anti-MTP (BD Sciences), secondary antibody peroxidase conjugate goat anti-rabbit (Chemicon International), anti-mouse IgG (Sigma-Aldrich), and donkey anti-goat (Santa Cruz Biotechnology) were from commercial sources.

Techniques: Immunostaining, Purification, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Luminal Lipid Regulates CD36 Levels and Downstream Signaling to Stimulate Chylomicron Synthesis *

doi: 10.1074/jbc.M111.233551

Figure Lengend Snippet: CD36 removal from BBM does not quantitatively match its intracellular transfer. CD36 content in purified BBM and jejunal mucosa from fasted control rats (C) and from rats 6 h after force-feeding with 3 ml of oil were assayed by both ELISA (A, B, D, and E) and Western blotting (C and F). To determine whether the oil effect on CD36 levels in BBM and jejunal mucosa is specific, a set of animals were also force-fed with a sucrose solution providing a similar caloric load as the oil gavage. The activity of sucrase, a BBM enzyme marker, was determined to verify the efficiency of BBM purification (B and E). The values are the means ± S.E. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti-mouse CD36 (R & D System or Santa Cruz Biotechnology), mouse anti-ubiquitin (Zymed Laboratories Inc., Invitrogen), anti-phospho-ERK1/2 (Thr-202/Tyr-204) and anti-ERK1/2 (Cell Signaling Technology), anti-apoB, anti-HSC70 (Santa Cruz Biotechnology), anti-MTP (BD Sciences), secondary antibody peroxidase conjugate goat anti-rabbit (Chemicon International), anti-mouse IgG (Sigma-Aldrich), and donkey anti-goat (Santa Cruz Biotechnology) were from commercial sources.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay, Marker

Journal: The Journal of Biological Chemistry

Article Title: Luminal Lipid Regulates CD36 Levels and Downstream Signaling to Stimulate Chylomicron Synthesis *

doi: 10.1074/jbc.M111.233551

Figure Lengend Snippet: Time course of lipid-induced drop in jejunal CD36 protein content. A and B, CD36 content in purified BBM (A) and jejunal mucosa (B) from fasted and oil gavaged rats was assayed by ELISA at 1, 4, 6, 9, and 12 h after the gavage. C, expression of CD36 in mice mucosa was analyzed by Western blotting (15 μg) at 1 and 4 h after force-feeding with oil and compared with fasted controls. D, to explore the specificity of the oil effect on CD36, expression of MTP was also examined. These data were normalized to HSC70 expression, which remained unchanged in these experimental conditions and served as an internal control for protein loading. The values are the means ± S.E. (n = 3). *, p < 0.05; **, p < 0.01. E and F, CD36 mRNA levels were assayed in jejunal mucosa from fasted control rats and experimental animals at 1, 4, 6, 12, and 30 h after force-feeding with 3 ml of sunflower oil (E) and in jejunal mucosa from fasted and experimental mice at 1 or 4 h after force-feeding with 0.5 ml of oil (F). The data were normalized to 18 S rRNA for differences in RNA loading. The values are the means ± S.E. (n = 5). **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti-mouse CD36 (R & D System or Santa Cruz Biotechnology), mouse anti-ubiquitin (Zymed Laboratories Inc., Invitrogen), anti-phospho-ERK1/2 (Thr-202/Tyr-204) and anti-ERK1/2 (Cell Signaling Technology), anti-apoB, anti-HSC70 (Santa Cruz Biotechnology), anti-MTP (BD Sciences), secondary antibody peroxidase conjugate goat anti-rabbit (Chemicon International), anti-mouse IgG (Sigma-Aldrich), and donkey anti-goat (Santa Cruz Biotechnology) were from commercial sources.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Luminal Lipid Regulates CD36 Levels and Downstream Signaling to Stimulate Chylomicron Synthesis *

doi: 10.1074/jbc.M111.233551

Figure Lengend Snippet: Lipids induce polyubiquitination of jejunal CD36 in vivo and in CD36-transfected CHO cells in vitro. A, fasted control mice were force-fed with 0.5 ml of oil and sacrificed 2 or 4 h later. The jejunal mucosa homogenates were immunoprecipitated (IP) with anti-CD36 antibody. Immunocomplexes were analyzed by Western blotting (WB) with anti-ubiquitin or anti-CD36 antibodies. The data are representative of three different experiments. B, CHO cells transfected with an empty vector (lane 1), FLAG-CD36 (lane 2), or double mutated CD36 (K469A and K472A) (lane 3) were treated 2 h with (100 μm) of the indicated lipid; oleic acid, 1–2 diolein, and 2-monolein were added in presence of BSA. The cells were lysed, and clarified lysates were immunoprecipitated (IP) with FLAG affinity gel. The immunocomplexes were then analyzed by Western blotting (WB) using an anti-ubiquitin antibody or an anti-CD36 antibody.

Article Snippet: Anti-mouse CD36 (R & D System or Santa Cruz Biotechnology), mouse anti-ubiquitin (Zymed Laboratories Inc., Invitrogen), anti-phospho-ERK1/2 (Thr-202/Tyr-204) and anti-ERK1/2 (Cell Signaling Technology), anti-apoB, anti-HSC70 (Santa Cruz Biotechnology), anti-MTP (BD Sciences), secondary antibody peroxidase conjugate goat anti-rabbit (Chemicon International), anti-mouse IgG (Sigma-Aldrich), and donkey anti-goat (Santa Cruz Biotechnology) were from commercial sources.

Techniques: In Vivo, Transfection, In Vitro, Immunoprecipitation, Western Blot, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Luminal Lipid Regulates CD36 Levels and Downstream Signaling to Stimulate Chylomicron Synthesis *

doi: 10.1074/jbc.M111.233551

Figure Lengend Snippet: Degradation of CD36 by the proteasome (A and B) leads to a decrease in the ERK1/2 (A and C) activation in CD36+/+ mice but not in CD36−/− mice. Control fasted CD36+/+ and CD36−/− mice were injected intraperitoneally with MG132 (14 mg/kg) 30 min before gavage (0.5 ml oil) and sacrificed 4 h later. CD36, HSC70, phosphorylated ERK1/2 (P-ERK1/2), and ERK1/2 levels in jejunal mucosa were analyzed by Western blotting. A representative signal is shown from four independent experiments, quantified by densitometry, and standardized to HSC70 signal or total ERK1/2 as the loading control. The data are expressed as percentages of controls not treated with MG132. The values are the means ± S.E. (n = 4). *, p < 0.05.

Article Snippet: Anti-mouse CD36 (R & D System or Santa Cruz Biotechnology), mouse anti-ubiquitin (Zymed Laboratories Inc., Invitrogen), anti-phospho-ERK1/2 (Thr-202/Tyr-204) and anti-ERK1/2 (Cell Signaling Technology), anti-apoB, anti-HSC70 (Santa Cruz Biotechnology), anti-MTP (BD Sciences), secondary antibody peroxidase conjugate goat anti-rabbit (Chemicon International), anti-mouse IgG (Sigma-Aldrich), and donkey anti-goat (Santa Cruz Biotechnology) were from commercial sources.

Techniques: Activation Assay, Injection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Luminal Lipid Regulates CD36 Levels and Downstream Signaling to Stimulate Chylomicron Synthesis *

doi: 10.1074/jbc.M111.233551

Figure Lengend Snippet: The LCFA-mediated ERK1/2 activation requires CD36 and triggers an up-regulation of apoB48 and MTP protein expression. Isolated intestinal segments from CD36+/+ and CD36−/− mice were cultured in the presence of linoleic acid (240 μm, complexed with BSA) for 10–20 min. A, CD36, phosphorylated ERK1/2 (P-ERK1/2), and ERK1/2 levels in jejunal mucosa were analyzed by Western blotting. A representative signal is shown from four independent experiments, quantified by densitometry, and standardized to total ERK1/2 as the loading control. B, apoB48, MTP, and HSC70 levels in jejunal mucosa were analyzed by Western blotting. A representative signal is shown from four independent experiments, quantified by densitometry, and standardized to HSC70 as the loading control. The data are expressed as percentages of controls not treated with LCFA. The values are the means ± S.E. (n = 4). *, p < 0.05; **, p < 0.01.

Article Snippet: Anti-mouse CD36 (R & D System or Santa Cruz Biotechnology), mouse anti-ubiquitin (Zymed Laboratories Inc., Invitrogen), anti-phospho-ERK1/2 (Thr-202/Tyr-204) and anti-ERK1/2 (Cell Signaling Technology), anti-apoB, anti-HSC70 (Santa Cruz Biotechnology), anti-MTP (BD Sciences), secondary antibody peroxidase conjugate goat anti-rabbit (Chemicon International), anti-mouse IgG (Sigma-Aldrich), and donkey anti-goat (Santa Cruz Biotechnology) were from commercial sources.

Techniques: Activation Assay, Expressing, Isolation, Cell Culture, Western Blot

Journal: PLoS Pathogens

Article Title: Nrf2, a PPARγ Alternative Pathway to Promote CD36 Expression on Inflammatory Macrophages: Implication for Malaria

doi: 10.1371/journal.ppat.1002254

Figure Lengend Snippet: (A–B) CD36 protein and mRNA levels detected by flow cytometry or qRT-PCR on Swiss murine peritoneal macrophages firstly treated during 24 h with TNF-α (10 ng/mL), peptidoglycan (PGN) (1 µg/mL) or Plasmodium falciparum culture supernatant ( P.f. c s) and incubated during 20 supplementary hours with rosiglitazone (R) (5 µM) or IL13 (50 ng/mL) for protein quantification or 5 supplementary hours for mRNA detection. Data are represented as a mean ± SD of three independent experiments. **p<0.01 compared with control cells (untreated). (C) CD36 protein level detected by flow cytometry on macrophages pre-incubated with a TL2 blocking monoclonal antibody (Anti-TLR2 mAb) (10 µg/mL) or Etanercept, a TNF-α inhibitor (10 µg/mL), and stimulated with TNF-α (10 ng/mL), PGN (1 µg/mL) or P.f . cs. Data are from a representative experiment performed in triplicate ± SD. **p<0.01 compared with control cells (untreated). ## p<0.01 compared with the respective control (Etanercept treated cells). (D) Phagocytosis index of P. falciparum unopsonized erythrocytes by Swiss murine macrophages stimulated as described in (A). Data are from a representative experiment performed in triplicate ± SD. The experiment was repeated three times. *p<0.05 and **p<0.01 compared with control cells (untreated). (E) PPARγ protein and mRNA levels in Swiss macrophages were determined by qRT-PCR after treatment of cells with TNF-α, PGN or P.f. cs. Data are represented as a mean ± SD of three independent experiments for mRNA quantification. **p<0.01 compared with control cells (C).

Article Snippet: Briefly, murine macrophage CD36 expression was detected using a PE-monoclonal CD36 antibody (Santacruz, sc-13572) and compared with an irrelevant appropriate isotype control (Santa Cruz, sc-3600). hMDMs macrophages were stained with mouse IgM,κ CD36-APC antibody (BD Pharmingen).

Techniques: Flow Cytometry, Quantitative RT-PCR, Incubation, Control, Blocking Assay

Journal: PLoS Pathogens

Article Title: Nrf2, a PPARγ Alternative Pathway to Promote CD36 Expression on Inflammatory Macrophages: Implication for Malaria

doi: 10.1371/journal.ppat.1002254

Figure Lengend Snippet: (A–B) CD36 protein and mRNA levels detected by flow cytometry or qRT-PCR on Swiss murine peritoneal macrophages firstly treated during 24 h with TNF-α (10 ng/mL), PGN (1 µg/mL) or P. falciparum culture ( P.f. c s) and then incubated during 20 h with sulforaphane (SFN) (10 µM) or diethylmaleate (DEM) (100 µM) for protein quantification or 5 supplementary hours for mRNA detection. Data are represented as a mean ± SD of three independent experiments. **p<0.01 compared with the respective control (untreated); ## p<0.01 compared with the respective control (TNF-α treated cells); ¤¤ p<0.01 compared with the respective control (PGN treated cells); §§ p<0.01 compared with the respective control ( P.f . cs treated cells); δδ p<0.01compared with control SFN-treated cells; ¶¶ p<0.01compared to control DEM-treated cells. (C) Phagocytosis index of Pf PEs by Swiss murine macrophages stimulated as described in (A). Data are represented as a mean ± SD of three independent experiments. *p<0.05 and **p<0.01 compared with the respective control (untreated); ## p<0.01 compared with the respective control (TNF-α treated cells); $$ p<0.01 compared with the respective control (SFN treated cells); ££ p<0.01 compared with the respective control (TNF+SFN treated cells) (D) HO-1 mRNA level in Swiss peritoneal macrophages determined by qRT-PCR after treatment of cells during 24 h with TNF-α (10 ng/mL) and then during 5 h with rosiglitazone (R) (5 µM), IL13 (50 ng/mL) or SFN (10 µM). Data are represented as a mean ± SD of three independent experiments. **p<0.01 compared with the respective control (untreated); ## p<0.01 compared with the respective control (TNF-α treated cells).

Article Snippet: Briefly, murine macrophage CD36 expression was detected using a PE-monoclonal CD36 antibody (Santacruz, sc-13572) and compared with an irrelevant appropriate isotype control (Santa Cruz, sc-3600). hMDMs macrophages were stained with mouse IgM,κ CD36-APC antibody (BD Pharmingen).

Techniques: Flow Cytometry, Quantitative RT-PCR, Incubation, Control

Journal: PLoS Pathogens

Article Title: Nrf2, a PPARγ Alternative Pathway to Promote CD36 Expression on Inflammatory Macrophages: Implication for Malaria

doi: 10.1371/journal.ppat.1002254

Figure Lengend Snippet: (A) PPARγ protein level on Swiss murine peritoneal macrophages and on RAW264.7 cells. The experiments were repeated three times. (B) CD36 protein level on murine RAW264.7 cells after treatment of cells with IL13 (50 ng/mL), rosiglitazone (R) (5 µM), sulforaphane (SFN) (10 µM) or diethylmaleate (DEM) (100 µM). Data are represented as a mean ± SD of three independent experiments. **p<0.01 compared with control cells. (C) CD36 protein level detected by flow cytometry on Swiss murine peritoneal macrophages firstly treated during 1 h with the PPARγ antagonists GW9662 (5 µM) and T007 (2 µM) and then incubated during 20 h with IL13, SFN or DEM. Data are represented as a mean ± SD of three independent experiments. **p<0.01 compared with the respective control (untreated); ## p<0.01 compared with the respective control (GW9662 treated cells); §§ p<0.01 compared with the respective control (T007 treated cells). (D) CD36 mRNA level on PPARγ +/+ and PPARγ −/− C57BL/6 murine peritoneal macrophages after treatment of cells with rosiglitazone, IL13, SFN or DEM. Data are from a representative experiment performed in triplicate ± SD. The experiment was repeated three times. *p<0.05 and **p<0.01 compared with the respective control (PPARγ +/+ ); ## p<0.01 compared with the respective control (PPARγ −/− ). (E) Phagocytosis index of P. falciparum unopsonized erythrocytes by murine PPARγ +/+ and PPARγ −/− C57BL/6 macrophages stimulated as described in (E). Data are from a representative experiment performed in triplicate ± SD. The experiment was repeated three times. *p<0.05 and **p<0.01 compared with the respective control (PPARγ +/+ ); # p<0.05 compared with the respective control (PPARγ −/− ).

Article Snippet: Briefly, murine macrophage CD36 expression was detected using a PE-monoclonal CD36 antibody (Santacruz, sc-13572) and compared with an irrelevant appropriate isotype control (Santa Cruz, sc-3600). hMDMs macrophages were stained with mouse IgM,κ CD36-APC antibody (BD Pharmingen).

Techniques: Control, Flow Cytometry, Incubation

Journal: PLoS Pathogens

Article Title: Nrf2, a PPARγ Alternative Pathway to Promote CD36 Expression on Inflammatory Macrophages: Implication for Malaria

doi: 10.1371/journal.ppat.1002254

Figure Lengend Snippet: CD36 mRNA level on Swiss peritoneal macrophages treated during 24 h with TNF-α (10 ng/mL) (A) and on RAW264.7 cells (B) and transfected with siRNA targeting Nrf2 (siRNA Nrf2) or control siRNA (siRNA control) and stimulated with sulforaphane SFN (10 µM) or diethylmaleate (DEM) (100 µM). Data are represented as a mean ± SD of three independent experiments. *p<0.05 and **p<0.01 compared with the respective control (cells transfected with siRNA control), (C) CD36 mRNA level on Nrf2 +/+ and Nrf2 −/− C57BL/6 murine peritoneal macrophages after treatment during 24 h with TNF-α and then incubated during 5 h with SFN or DEM. Data are from a representative experiment performed in triplicate ± SD. The experiment was repeated three times **p<0.01 compared with the respective control (Nrf2 +/+ ), ## p<0.01 compared with the respective control (Nrf2 +/+ cells treated with TNF-α). (D) Phagocytosis index of Pf PEs by murine Nrf2 +/+ and Nrf2 −/− C57BL/6 macrophages stimulated as described in (C). Data are from a representative experiment performed in triplicate ± SD. The experiment was repeated three times. *p<0.05 and **p<0.01 compared with the respective control (Nrf2 +/+ control cells); # p<0.05 compared with the respective control (Nrf2 +/+ cells treated with TNF-α).

Article Snippet: Briefly, murine macrophage CD36 expression was detected using a PE-monoclonal CD36 antibody (Santacruz, sc-13572) and compared with an irrelevant appropriate isotype control (Santa Cruz, sc-3600). hMDMs macrophages were stained with mouse IgM,κ CD36-APC antibody (BD Pharmingen).

Techniques: Transfection, Control, Incubation

Journal: PLoS Pathogens

Article Title: Nrf2, a PPARγ Alternative Pathway to Promote CD36 Expression on Inflammatory Macrophages: Implication for Malaria

doi: 10.1371/journal.ppat.1002254

Figure Lengend Snippet: (A) CD36 protein level detected by flow cytometry on human-monocytes derived macrophages (hMDMs) firstly treated during 24 h with TNF-α (10 ng/mL) or PGN (1 µg/mL) and then incubated during 20 h with rosiglitazone (R) (5 µM), IL13 (50 ng/mL), SFN (10 µM) or DEM (100 µM). Data are represented as a mean ± SD of three independent experiments. **p<0.01 compared with the respective control (untreated); # p<0.05 compared with the respective control (TNF-α treated cells); ¤¤ p<0.01 compared with the respective control (PGN treated cells). (B) PPARγ protein and mRNA levels on hMDMs treated with TNF-α, LPS, P.f . cs and PGN. PPARγ western blot is representative of three independent experiments and qPCR data are represented as a mean ± SD of three independent experiments. **p<0.01 compared with the respective control (untreated). (C) Phagocytosis index of P. falciparum unopsonized erythrocytes by hMDMs stimulated as described in (A). Data are represented as a mean ± SD of three independent experiments. **p<0.01 compared with the respective control (untreated); ## p<0.01 compared with the respective control (PGN treated cells); $$ p<0.01 compared with the respective control (SFN treated cells); ££ p<0.01 compared with the respective control (TNF+SFN treated cells).

Article Snippet: Briefly, murine macrophage CD36 expression was detected using a PE-monoclonal CD36 antibody (Santacruz, sc-13572) and compared with an irrelevant appropriate isotype control (Santa Cruz, sc-3600). hMDMs macrophages were stained with mouse IgM,κ CD36-APC antibody (BD Pharmingen).

Techniques: Flow Cytometry, Derivative Assay, Incubation, Control, Western Blot

Journal: PLoS Pathogens

Article Title: Nrf2, a PPARγ Alternative Pathway to Promote CD36 Expression on Inflammatory Macrophages: Implication for Malaria

doi: 10.1371/journal.ppat.1002254

Figure Lengend Snippet: Swiss mice receiving PGN (200 µg/mouse) (subcutaneous route) two days before infection and d,L-sulforaphane (75 mg/kg) or rosiglitazone (3 mg/kg) (oral route) during five days postinfection were infected with 1×10 6 Plasmodium berghei parasites via intra-peritoneal injection. (A) and (C) Survival was assessed twice daily. **p<0.01 compared with untreated mice (control), ¤¤ p<0.01 compared with PGN-treated mice. (B) and (D) Parasitemia levels were assessed daily. **p<0.01 compared with untreated macrophages (control); ¤ p<0.05 and ¤¤ p<0.01 compared with PGN-treated cells. (E) and (F) CD36 expression on macrophages was measured the day of infection by flow cytometry on three independent mice. *p<0.05 compared with untreated macrophages (control); ¤ p<0.05 compared with PGN-treated cells. (G) Phagocytosis index of P. berghei unopsonized erythrocytes by Swiss murine macrophages incubated with FcR blocking antibodies (20 µg/mL) and CD36 blocking antibodies (10 µg/mL). Data are presented as mean ± SD of one experiment performed in triplicate. **p<0.01 compared to uRBC+α−FcR, $ p<0.05 compared with iRBC+α−CD36, £ p<0.05 compared with iRBC+α−FcR. (H) Phagocytosis index of P. berghei unopsonized erythrocytes by 3 days-infected macrophages from Swiss mice treated with PGN and SFN as described above. ¤ p<0.05 compared with PGN-treated macrophages.

Article Snippet: Briefly, murine macrophage CD36 expression was detected using a PE-monoclonal CD36 antibody (Santacruz, sc-13572) and compared with an irrelevant appropriate isotype control (Santa Cruz, sc-3600). hMDMs macrophages were stained with mouse IgM,κ CD36-APC antibody (BD Pharmingen).

Techniques: Infection, Injection, Control, Expressing, Flow Cytometry, Incubation, Blocking Assay

Journal: PLoS ONE

Article Title: Reduction of the HIV Protease Inhibitor-Induced ER Stress and Inflammatory Response by Raltegravir in Macrophages

doi: 10.1371/journal.pone.0090856

Figure Lengend Snippet: Real time PCR primers.

Article Snippet: Mouse monoclonal antibody against CD36 was from Cayman Chemical (Ann Arbor, MI).

Techniques: Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Reduction of the HIV Protease Inhibitor-Induced ER Stress and Inflammatory Response by Raltegravir in Macrophages

doi: 10.1371/journal.pone.0090856

Figure Lengend Snippet: ( A–C ) Human THP-1-derived macrophages were treated with HIV PIs (lopinavir/Ritonavir, LOPV/RITV, 25 µM) with or without raltegravir (25 µM) for 24 h. The total cellular RNA was isolated and reverse transcribed. The relative mRNA levels of CD36, SRA, and LDLR were determined by real-time PCR as described in “Methods”. The values are means ± S.E. of three independent experiments. **, p<0.01, ***, p<0.001, statistical significance relative to vehicle control. #, p<0.05, statistical significance of HIV PI+raltegravir-treated group relative to HIV PI-treated group. ( D ) Human THP-1-derived macrophages were treated with HIV PIs (lopinavir/Ritonavir, LOPV/RITV, 25 µM) with or without raltegravir (25 µM) for 24 h. The total protein lysates were prepared and subjected to Western blot analysis. Representative immunoblots for CD36, SRA and β-actin are shown. β-actin was used as the loading control for total proteins.

Article Snippet: Mouse monoclonal antibody against CD36 was from Cayman Chemical (Ann Arbor, MI).

Techniques: Derivative Assay, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Western Blot