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: Cancers

Article Title: Murlentamab, a Low Fucosylated Anti-Müllerian Hormone Type II Receptor (AMHRII) Antibody, Exhibits Anti-Tumor Activity through Tumor-Associated Macrophage Reprogrammation and T Cell Activation

doi: 10.3390/cancers13081845

Figure Lengend Snippet: Flow cytometry antibodies used.

Article Snippet: CD36-PEVio770 , Miltenyi , 130-110-742 , REA770 , .

Techniques: Flow Cytometry, In Vivo, In Vitro

Journal: Cancers

Article Title: Murlentamab, a Low Fucosylated Anti-Müllerian Hormone Type II Receptor (AMHRII) Antibody, Exhibits Anti-Tumor Activity through Tumor-Associated Macrophage Reprogrammation and T Cell Activation

doi: 10.3390/cancers13081845

Figure Lengend Snippet: Murlentamab opsonization of SKOV3-R2 + orients naïve macrophages and reprograms TAMs towards an M1-like profile. SKOV3-R2 + ovarian tumor cells were labeled with different 3C23K antibodies (3C23K-FcKO control, 3C23K-CHO normally fucosylated or murlentamab the low fucosylated form) and cultured in the presence of human monocyte-derived macrophages from healthy donors unstimulated (M0) or stimulated with M-CSF and IL-10 (TAMs). ( A ) The proportion of macrophages expressing M1/M2 membrane markers (CD32, CD64, CD80, TLR2, CD163, CD36 and CD206) was determined by flow cytometry after three days of co-culture with SKOV3-R2 + cells. ( B ) The release of cytokines (IL1β, IL12, TNFα, IL6, IFNγ, IL10) and chemokines (CCL2, CCL4, CCL5, CXCL9 and CXCL10) in the culture medium was determined by AlphaLISA after three days of co-culture with SKOV3-R2 + cells. Data shown (boxplots) are the results from three different experiments (performed with three different healthy donors). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. p values were determined using one-way ANOVA analysis followed by Tukey’s multiple comparisons test.

Article Snippet: CD36-PEVio770 , Miltenyi , 130-110-742 , REA770 , .

Techniques: Labeling, Cell Culture, Derivative Assay, Expressing, Membrane, Flow Cytometry, Co-Culture Assay

Journal: The Journal of Biological Chemistry

Article Title: Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36

doi: 10.1016/j.jbc.2021.101311

Figure Lengend Snippet: High CD36 expression in S508A mutant and effect of CD36 RNAi. A – F , staining of CD36 ( green ) in HepG2 cell lines. G , quantitation of CD36 staining for the six cell lines (see ). H – J , S508A mutants stained with CD36 ( green ), F-actin ( red ), and DAPI ( blue ). CD36 and BC expression of untreated ( H ) and RNAi control-treated ( I ) S508A mutant cells versus RNAi to CD36-treated S508A mutant cells ( J ). BCs are shown with arrows . K – N , lipid droplet staining ( green ) of S508A mutant cells. Control RNAi treatment at 20× ( K ) and 40× ( L ). RNAi to CD36 treatment at 20× ( M ) and 40× ( N ). O , mRNA expression levels in cell lines by qRT-PCR (in triplicate, relative to GAPDH). P , treatment of cell lines with no RNAi, control RNAi, RNAi-1, RNAi-2, and RNAi-1 plus RNAi-2 to CD36 (in triplicate, relative to GAPDH). Q , cell surface levels of CD36 on four cell lines. R , SDS gel analysis of CD36 expression in six cell lines. KO, knockout; SA, S508A mutant; SD, S508D mutant; WT, wild type.

Article Snippet: Goat anti-human CD36 (50 μg; R&D, AF1955) was immobilized on a Thermo Scientific IP column, 26149, washed 3× with lysis buffer, incubated with cell lysates, washed 3× with lysate buffer, and eluted with 2× SDS sample buffer.

Techniques: Expressing, Mutagenesis, Staining, Quantitation Assay, Quantitative RT-PCR, SDS-Gel, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36

doi: 10.1016/j.jbc.2021.101311

Figure Lengend Snippet: Nuclear expression of LKB1 in WT, CEACAM1 −/− , and S508A and S508D mutants and AMPK expression. LKB1 nuclear expression in WT ( A ) and in CEACAM1 −/− ( B ) and in S508A ( C ) and S508D ( D ) mutants ( red = LKB1, blue = DAPI). Percent LKB1 positive nuclei per 50 cells counted for three fields ±SEM shown underneath each panel. Magnification 20×. E , triple staining of S508A mutant for CD36 ( green ), LKB1 ( red ) and nuclei ( blue ). Magnification 40×. F , immunoblots for the detection of activated AMPK ( green channel ) in WT, CEACAM1 KO, and CEACAM1 mutants (tubulin in red channel ). Twenty micrograms of protein lysate loaded per lane.

Article Snippet: Goat anti-human CD36 (50 μg; R&D, AF1955) was immobilized on a Thermo Scientific IP column, 26149, washed 3× with lysis buffer, incubated with cell lysates, washed 3× with lysate buffer, and eluted with 2× SDS sample buffer.

Techniques: Expressing, Staining, Mutagenesis, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36

doi: 10.1016/j.jbc.2021.101311

Figure Lengend Snippet: PCSK9 expression affects CD36 expression in the S508A mutant. PCSK9 expression in WT ( A ) and in CEACAM1 S508A mutant ( B ) HepG2 cells. Triple stained for PCSK9 ( green ), F-actin ( red ), and nuclei ( blue ). Effect of control RNAi ( C ) and PCSK9 RNAi ( D ) on CD36 expression ( green ) in CEACAM1 S508A mutant HepG2 cells. Lack of an effect of control RNAi ( E ) and CD36 RNAi ( F ) on PCSK9 expression ( green ). G , effect of PCSK9 RNAi on PCSK9 mRNA expression as measured by qRT-PCR. H , effect of PCSK9 RNAi on CD36 mRNA expression as measured by qRT-PCR in triplicate.

Article Snippet: Goat anti-human CD36 (50 μg; R&D, AF1955) was immobilized on a Thermo Scientific IP column, 26149, washed 3× with lysis buffer, incubated with cell lysates, washed 3× with lysate buffer, and eluted with 2× SDS sample buffer.

Techniques: Expressing, Mutagenesis, Staining, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36

doi: 10.1016/j.jbc.2021.101311

Figure Lengend Snippet: Tyrosine phosphorylation of CEACAM1 by insulin versus Src and coexpression of SRC and CD36 with CEACAM1 in mutant S508A cells. A , lysates (20 μg) from WT cells treated before and after with insulin (15 μg/ml) over time were IPed with anti-CEACAM1 antibody and immunoblotted for phosphotyrosine. B , lysates (20 μg) from cells treated before and after with insulin for 30 min were IPed with anti-CEACAM1 antibody and immunoblotted for phosphotyrosine (normalized for CEACAM1 signal in a parallel immunoblot). C and D , equal amounts of lysates from S508A ( C ) or S508D ( D ) mutants treated with insulin or inhibitors of Src (BOS, 10 μM), PI3K (LY294002, 20 μM), or CaMK2 (KN93, 20 μM), IPed with anti-CEACAM1 antibody and run on SDS gels were blotted for phosphotyrosine. Mutant S508A cells were stained for Src ( red ), CEACAM1 ( green ), DAPI ( blue ), and overlayed ( E ) or for Src ( red ), CD36 ( green ), DAPI ( blue ), and overlayed ( F ). Arrows indicate examples of prominent BCs with yellow staining indicating overlap of Src with CEACAM1 or CD36. Magnifications were 40× ( E ) and 20× ( F ).

Article Snippet: Goat anti-human CD36 (50 μg; R&D, AF1955) was immobilized on a Thermo Scientific IP column, 26149, washed 3× with lysis buffer, incubated with cell lysates, washed 3× with lysate buffer, and eluted with 2× SDS sample buffer.

Techniques: Mutagenesis, Western Blot, Staining

Journal: The Journal of Biological Chemistry

Article Title: Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36

doi: 10.1016/j.jbc.2021.101311

Figure Lengend Snippet: Coimmunoprecipitation of CEACAM1, Src, LB1, and Annexin A2 with CD36. CD36 was immunoprecipitated from Ser508A mutant HepG2 cells, pretreated or not with insulin, run on SDS gels, and immunoblotted for CD36, CEACAM1, Src, LKB1, and Annexin A2 (AnxA2). Equal amounts of protein were probed on immunoblots.

Article Snippet: Goat anti-human CD36 (50 μg; R&D, AF1955) was immobilized on a Thermo Scientific IP column, 26149, washed 3× with lysis buffer, incubated with cell lysates, washed 3× with lysate buffer, and eluted with 2× SDS sample buffer.

Techniques: Immunoprecipitation, Mutagenesis, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36

doi: 10.1016/j.jbc.2021.101311

Figure Lengend Snippet: Model of FA import by CD36 and lipid droplet management in WT, CEACAM1 −/− , and CEACAM1 mutants in HepG2 cells. A , lipid droplets (LDs) increase in CEACAM1 −/− versus WT cells and are reduced in S508A mutant cells along with an increase in size of bile canaliculi (BCs). B , CD36 is found in a complex with CEACAM1, Src, LKB1, and AMPK. Src can phosphorylate CEACAM1 on Y493 and Y520 located on either side of S508. LKB1 can phosphorylate AMPK; however, phosphorylated LKB1 can translocate to the nucleus, reducing phosphorylation of AMPK. CD36 can be internalized to endosomes after binding long chain fatty acids and shunted to either lysosomes for degradation or to BCs. C , phosphorylation of S512 by PKA followed by phosphorylation of S508 by GSK3β can lead to high lipid accumulation. Src phosphorylation of Y493 leads to high lipid accumulation, while phosphorylation of Y520 leads to low lipid accumulation. Phosphorylation of both tyrosines is influenced by the phosphorylation status of S508. Thus, the null mutants of Y493 and Y520 can abrogate their normal lipid regulation functions.

Article Snippet: Goat anti-human CD36 (50 μg; R&D, AF1955) was immobilized on a Thermo Scientific IP column, 26149, washed 3× with lysis buffer, incubated with cell lysates, washed 3× with lysate buffer, and eluted with 2× SDS sample buffer.

Techniques: Mutagenesis, Binding Assay

Journal: Redox Biology

Article Title: Radiosensitizing capacity of fenofibrate in glioblastoma cells depends on lipid metabolism

doi: 10.1016/j.redox.2024.103452

Figure Lengend Snippet: Fenofibrate (FF) modulates lipid droplets (LDs) in U87 and LN18 cells. A) Schematic representation of uptake and processing pathways of fatty acids (FAs) in tumor cells. B-i) Percentage of U87 and LN18 cells expressing CD36. B-ii) Mean fluorescent intensity (MFI) of CD36 expression in U87 and LN18 cells. C) U87 and LN18 cells were stained with LipidTOX™ (red) for LD, GPAT4 antibody (green) for lipid enzyme and Hoechst (blue) for nuclei. D) Representative immunoblots and bar graphs (from 3 independent experiments) of basal GPAT4 (D-i) and DGAT1 (D-ii) expression in U87 and LN18 cells. E) Representative confocal images of LDs (red) and nuclei (blue) of U87 and LN18 cells for all treatment groups. F) Graph showing numbers of small and large LDs in U87 (top) and LN18 (bottom) cells per treatment. LD quantification was performed by counting red lipid bodies using ImageJ (minimum of 50 cells per cell line). G) Representative immunoblots and bar graphs showing the expression of DGAT1 in U87 (top) and LN18 (bottom) cells after FF and RTx treatment. H) The normalized value of intracellular GPAT4 in U87 (top) and LN18 (bottom) cells in all treatment groups. Unless stated otherwise, data are shown as means ± SD of at least three independent experiments. P values were calculated using an unpaired t -test (two groups) or a two-way analysis of variance (ANOVA, more than two groups) with Tukey's correction. ∗p < 0.0332, ∗∗p < 0.0021, ∗∗∗p < 0.0002 and ∗∗∗∗p < 0.0001.

Article Snippet: Cells (0.25x10 6 ) were trypsinized, washed twice with ice-cold flow cytometry buffer (phosphate-buffered saline (PBS; Life Technologies)), containing 10 % v/v FBS (Sigma-Aldrich) and incubated with FITC-conjugated CD36 monoclonal antibody (mAb, AC106, 2 μg/mL, Miltenyi Biotec), and membrane Hsp70 mAb (cmHsp70.1, 20 μg/mL, multimmune GmbH) for 30 min in the dark on ice.

Techniques: Expressing, Staining, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: SIRT3 deficiency exacerbates fatty liver by attenuating the HIF1α-LIPIN 1 pathway and increasing CD36 through Nrf2

doi: 10.1186/s12964-020-00640-8

Figure Lengend Snippet: Enhanced hepatic VLDLR and CD36 levels in Sirt3 -deficient mice fed a HFD. mRNA abundance ( a ) and protein levels ( b ) of VLDLR in livers of WT and Sirt3 −/− mice fed either a standard chow or a HFD. CD36 ( c ), total and phospho-Nrf2 ( d ), Keap1 ( e ) and NQO1 ( f ) protein levels. g , ROS levels. h , PPARγ protein levels. Data are presented as the mean ± S.D. ( n = 6 per group). a, p < 0.05 vs. WT mice fed a standard chow. b, p < 0.05 vs. WT mice fed a HFD. c, p < 0.05 vs. Sirt3 −/− mice fed a standard chow

Article Snippet: Western blot analysis was performed using antibodies against HIF1α (sc-10,790), LIPIN1 (sc-98,450), Histone H3 (sc-10,809), SREBP1 (sc-365,513), Nrf2 (sc-722), NQO1 (sc-393,736), PPARγ (sc-7273) (Santa Cruz Biotechnology), SIRT3 (#5490), phospho-mTOR Ser2481 (#2974), mTOR (#2972), Keap1 (#4678 s) (Cell Signaling Technology Inc., Danvers, MA), GAPDH (MAB374) (Merck Millipore), CD36 (NB400–144) (Novus Biologicals, Centennial, CO), VLDLR (AF2258) (R&D Systems, Minneapolis, MN).

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: SIRT3 deficiency exacerbates fatty liver by attenuating the HIF1α-LIPIN 1 pathway and increasing CD36 through Nrf2

doi: 10.1186/s12964-020-00640-8

Figure Lengend Snippet: The increase in CD36 levels caused by lipids in Sirt3 -deficient hepatocytes is mediated by Nrf2. VLDLR mRNA abundance ( a ) and protein levels of VLDLR and NQO1, an Nrf-2-target gene, ( b ) were assessed in Huh-7 cells incubated with fatty acid free-BSA or BSA-palmitate (0.3 mM) and exposed to either vehicle or the Sirt3 inhibitor AAPBO (100 μM) for 16 h. a, p < 0.05 vs. CT. b, p < 0.05 vs. CT cells incubated with palmitate. c, p < 0.05 vs. CT cells treated with AAPBO. c , fatty acid uptake in Huh-7 cells incubated with fatty acid free-BSA or BSA-palmitate (0.3 mM) and exposed to either vehicle or the Sirt3 inhibitor AAPBO (100 μM) for 16 h was measured by the uptake of BODIPY-C16. a, p < 0.05 vs. CT. b, p < 0.05 vs. CT cells incubated with palmitate. c, p < 0.05 vs. CT cells treated with AAPBO. mRNA abundance ( d ) and protein levels of VLDLR ( e ) in Huh-7 cells transfected with control (CT) or SIRT3 siRNA and incubated in the presence or absence 0.3 mM palmitate (Pal) for 24 h. Protein levels of CD36 ( f ), NQO1 ( g ) and PPARγ ( h ) in Huh-7 cells transfected with control (CT) or SIRT3 siRNA and incubated in the presence or absence 0.3 mM palmitate (Pal) or the Nrf2 inhibitor ML385 (10 μM) for 24 h. a, p < 0.05 vs. CT siRNA cells. b, p < 0.05 vs. CT siRNA cells incubated with palmitate. c, p < 0.05 vs. SIRT3 siRNA cells. d, p < 0.05 vs. CT siRNA cells incubated with palmitate and ML385

Article Snippet: Western blot analysis was performed using antibodies against HIF1α (sc-10,790), LIPIN1 (sc-98,450), Histone H3 (sc-10,809), SREBP1 (sc-365,513), Nrf2 (sc-722), NQO1 (sc-393,736), PPARγ (sc-7273) (Santa Cruz Biotechnology), SIRT3 (#5490), phospho-mTOR Ser2481 (#2974), mTOR (#2972), Keap1 (#4678 s) (Cell Signaling Technology Inc., Danvers, MA), GAPDH (MAB374) (Merck Millipore), CD36 (NB400–144) (Novus Biologicals, Centennial, CO), VLDLR (AF2258) (R&D Systems, Minneapolis, MN).

Techniques: Incubation, Transfection, Control

Journal: Cell Communication and Signaling : CCS

Article Title: SIRT3 deficiency exacerbates fatty liver by attenuating the HIF1α-LIPIN 1 pathway and increasing CD36 through Nrf2

doi: 10.1186/s12964-020-00640-8

Figure Lengend Snippet: Potential new mechanisms by which Sirt3 deficiency promotes hepatic steatosis in mice fed a HFD. Exposure to a HFD reduces Sirt3 and contributes to triglyceride accumulation in the liver. However, hepatic lipid accumulation is attenuated by the activation of an adaptive mechanism involving an increase in nuclear HIF-1α and Lipin 1. Longer exposures to a HFD exacerbates Sirt3 decrease leading to higher uptake of lipids through an Nrf-2 mediated increase in CD36 and VLDLR. This results in a higher increase in fatty acid accumulation, which in turn reduces succinate levels, ultimately suppressing the adaptive increase in HIF-1α and Lipin 1. FAO: Fatty acid oxidation

Article Snippet: Western blot analysis was performed using antibodies against HIF1α (sc-10,790), LIPIN1 (sc-98,450), Histone H3 (sc-10,809), SREBP1 (sc-365,513), Nrf2 (sc-722), NQO1 (sc-393,736), PPARγ (sc-7273) (Santa Cruz Biotechnology), SIRT3 (#5490), phospho-mTOR Ser2481 (#2974), mTOR (#2972), Keap1 (#4678 s) (Cell Signaling Technology Inc., Danvers, MA), GAPDH (MAB374) (Merck Millipore), CD36 (NB400–144) (Novus Biologicals, Centennial, CO), VLDLR (AF2258) (R&D Systems, Minneapolis, MN).

Techniques: Activation Assay