Journal: PLoS ONE

Article Title: Identification of the ER-resident E3 ubiquitin ligase RNF145 as a novel LXR-regulated gene

doi: 10.1371/journal.pone.0172721

Figure Lengend Snippet: ( A ) Expression of Rnf145 was evaluated in the indicated mouse tissues by qPCR. Bars indicate mean ± SD (n = 3) ( B , C ) The indicated ( B ) human and ( C ) mouse cell lines and primary macrophages were cultured in lipoprotein-depletion medium for 16 hours and subsequently treated for 6 hours with 1μM GW3965 (GW). ( D ) THP1 cells were cultured in sterol depletion medium for 16 hrs and then treated with 1μM GW3965 (GW) and 100nM LG100268 (LG) for 6 hrs as indicated. Subsequently, ABCA1 and RNF145 expression was determined by qPCR and each bar and error represents the mean fold-change of ligand-treated cells over sterol-depleted cells ± SD (n≥3).

Article Snippet: Membranes were probed with the following antibodies: LDLR (Abcam, clone EP1553Y, 1:4000), tubulin (Sigma, clone DM1A, ascites fluid, 1:5000), ABCA1 (Novus Biologicals, NB400-105, 1:1000), FLAG (Sigma, clone M2, 1:1000), GFP (Santa Cruz sc-9996, 1:500), Myc (Santa Cruz 9E10, 1:3000), HA (Covance, clone 16B12, ascites fluid, 1:6000), HMGCR (rabbit polyserum was a kind gift from Dr. Peter Edwards, UCLA), SREBP2 (BD Biosciences, clone 1C6, 1:1000), SREBP1 (ThermoFisher, clone 2A4, 1:1000), actin (Merck Millipore, clone C4, 1:5000), V5 (Invitrogen, 46–0705, 1:3000), RNF145 (Abgent AP18281b, 1:8000), and ubiquitin (Enzo life Sciences, clone FK2, 1:1000).

Techniques: Expressing, Cell Culture

Journal: PLoS ONE

Article Title: Identification of the ER-resident E3 ubiquitin ligase RNF145 as a novel LXR-regulated gene

doi: 10.1371/journal.pone.0172721

Figure Lengend Snippet: ( A ) RAW264.7 macrophages were treated with 5μg/mL Actinomycin D (ActD) for the indicated time and expression of Abca1 and Rnf145 was determined by qPCR and plotted as mean ± SD relative to untreated cells (n = 3), ( B ) HepG2 and RAW264.7 cells were treated with 1μM GW3965 for 6 hours in the presence or absence of 5μg/ml actinomycinD for 4 hours, after which expression of the indicated genes was measured by qPCR. Bars indicate mean ± SD (n = 3) ( C , D ) THP1 macrophages were cultured in sterol-depletion medium for 16 hrs and then treated with ( C ) 1μM GW3965 (GW) for the indicated time, or ( D ) with the indicated concentration of GW3965 for 4 hrs. Subsequently, gene expression was evaluated qPCR and bars indicate mean ± SD (n = 3)

Article Snippet: Membranes were probed with the following antibodies: LDLR (Abcam, clone EP1553Y, 1:4000), tubulin (Sigma, clone DM1A, ascites fluid, 1:5000), ABCA1 (Novus Biologicals, NB400-105, 1:1000), FLAG (Sigma, clone M2, 1:1000), GFP (Santa Cruz sc-9996, 1:500), Myc (Santa Cruz 9E10, 1:3000), HA (Covance, clone 16B12, ascites fluid, 1:6000), HMGCR (rabbit polyserum was a kind gift from Dr. Peter Edwards, UCLA), SREBP2 (BD Biosciences, clone 1C6, 1:1000), SREBP1 (ThermoFisher, clone 2A4, 1:1000), actin (Merck Millipore, clone C4, 1:5000), V5 (Invitrogen, 46–0705, 1:3000), RNF145 (Abgent AP18281b, 1:8000), and ubiquitin (Enzo life Sciences, clone FK2, 1:1000).

Techniques: Expressing, Cell Culture, Concentration Assay, Gene Expression

Journal: PLoS ONE

Article Title: Identification of the ER-resident E3 ubiquitin ligase RNF145 as a novel LXR-regulated gene

doi: 10.1371/journal.pone.0172721

Figure Lengend Snippet: ( A ) LXR ChIP-seq experiments in human THP1 cells (GSE28319) and RAW macrophage-like cells (GSE50944) were analyzed and used to identify active LXREs within the Rnf145/Rnf145 loci, as graphically illustrated. ( B , C ) Genomic location of the identified LXREs. In bold, nucleotides that were mutated to disrupt LXR binding ( C ) A 1kb genomic region upstream of the transcriptional start site of hRNF145 was cloned into a pGL3basic. The putative LXRE was also mutated as indicated above. The empty, RNF145 WT , RNF145 MUT , and ABCA1 reporter plasmids were co-transfected with or without RXRα and LXRα expression plasmids in HEK 293T cells. 24 hours post-transfection the cells were treated with 1μM GW3965 (LXR) and 100nM LG100268 (RXR) for 24 hours and measured for luciferase signal (n≥3). ( D ) Cells were transfected with an empty or a tandem LXRE-containing pGL2 as in C . In all luciferase experiments the transfection efficiency was normalized to co-transfected Renilla luciferase. Bars report normalized chemiluminescence relative to untreated control ± SD (n = 3).

Article Snippet: Membranes were probed with the following antibodies: LDLR (Abcam, clone EP1553Y, 1:4000), tubulin (Sigma, clone DM1A, ascites fluid, 1:5000), ABCA1 (Novus Biologicals, NB400-105, 1:1000), FLAG (Sigma, clone M2, 1:1000), GFP (Santa Cruz sc-9996, 1:500), Myc (Santa Cruz 9E10, 1:3000), HA (Covance, clone 16B12, ascites fluid, 1:6000), HMGCR (rabbit polyserum was a kind gift from Dr. Peter Edwards, UCLA), SREBP2 (BD Biosciences, clone 1C6, 1:1000), SREBP1 (ThermoFisher, clone 2A4, 1:1000), actin (Merck Millipore, clone C4, 1:5000), V5 (Invitrogen, 46–0705, 1:3000), RNF145 (Abgent AP18281b, 1:8000), and ubiquitin (Enzo life Sciences, clone FK2, 1:1000).

Techniques: ChIP-sequencing, Binding Assay, Clone Assay, Transfection, Expressing, Luciferase, 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: 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

Journal: Cardiovascular research

Article Title: Beneficial effects of combinatorial micronutrition on body fat and atherosclerosis in mice.

doi: 10.1093/cvr/cvr146

Figure Lengend Snippet: Figure 2 Lipistase enhances muscle lipid utilization in LDLrKO mice. (A) Representative images of anti-CD36 immunostaining on paraffin-embedded sections of oxidative muscle (gastrocnemius) from mice after 10-month lipistase treatment vs. controls (bars ¼ 50 mm). (B) Oxygen consumption measured after the 10-month treatment (***P , 0.001). (C) Immunoblot quantification of the membrane-bound CD36 protein in muscle and mRNA expression of marker genes for fatty acid transport and oxidation measured in gastrocnemius 10 months after lipistase treatment (*P , 0.05).

Article Snippet: For CD36 quantification by immunoblotting, 4 mg and 6 mg of cytosolic and membrane extracts, respectively, were loaded and probed with 1/1000 rabbit polyclonal anti-CD36 antibody (Novus Biologicals, Inc. Littleton, CO, USA).

Techniques: Immunostaining, Western Blot, Membrane, Expressing, Marker

Journal: Circulation: Cardiovascular Genetics

Article Title: CD36 Is a Matrix Metalloproteinase-9 Substrate That Stimulates Neutrophil Apoptosis and Removal During Cardiac Remodeling

doi: 10.1161/circgenetics.115.001249

Figure Lengend Snippet: Figure 3: MMP-9 regulates CD36 levels through proteolytic degradation. (A) Full length CD36

Article Snippet: After blocking with 5% nonfat milk (Bio-Rad), the membrane was probed with primary antibodies: rabbit anti-CD36 polyclonal antibody (Novus BiologicalsNB400-144) at 1:1000, goat anti-CILP antibody (Aviva Systems Biology, OAEB01146) at 1:1000, or rabbit anti-caspase-3 (Cell Signaling, 9662) at

Techniques:

Journal: Circulation: Cardiovascular Genetics

Article Title: CD36 Is a Matrix Metalloproteinase-9 Substrate That Stimulates Neutrophil Apoptosis and Removal During Cardiac Remodeling

doi: 10.1161/circgenetics.115.001249

Figure Lengend Snippet: Figure 2: MMP-9 deletion increased CD36 and CILP levels at day 7 post-myocardial infarction

Article Snippet: After blocking with 5% nonfat milk (Bio-Rad), the membrane was probed with primary antibodies: rabbit anti-CD36 polyclonal antibody (Novus BiologicalsNB400-144) at 1:1000, goat anti-CILP antibody (Aviva Systems Biology, OAEB01146) at 1:1000, or rabbit anti-caspase-3 (Cell Signaling, 9662) at

Techniques:

Journal: Circulation: Cardiovascular Genetics

Article Title: CD36 Is a Matrix Metalloproteinase-9 Substrate That Stimulates Neutrophil Apoptosis and Removal During Cardiac Remodeling

doi: 10.1161/circgenetics.115.001249

Figure Lengend Snippet: Figure 5: MMP-9 regulates phagocytosis through CD36. As shown by the in vivo stimulated

Article Snippet: After blocking with 5% nonfat milk (Bio-Rad), the membrane was probed with primary antibodies: rabbit anti-CD36 polyclonal antibody (Novus BiologicalsNB400-144) at 1:1000, goat anti-CILP antibody (Aviva Systems Biology, OAEB01146) at 1:1000, or rabbit anti-caspase-3 (Cell Signaling, 9662) at

Techniques: In Vivo

Journal: Circulation: Cardiovascular Genetics

Article Title: CD36 Is a Matrix Metalloproteinase-9 Substrate That Stimulates Neutrophil Apoptosis and Removal During Cardiac Remodeling

doi: 10.1161/circgenetics.115.001249

Figure Lengend Snippet: Figure 8: Diagram depicting the roles of MMP-9 mediated CD36 degradation in LV remodeling

Article Snippet: After blocking with 5% nonfat milk (Bio-Rad), the membrane was probed with primary antibodies: rabbit anti-CD36 polyclonal antibody (Novus BiologicalsNB400-144) at 1:1000, goat anti-CILP antibody (Aviva Systems Biology, OAEB01146) at 1:1000, or rabbit anti-caspase-3 (Cell Signaling, 9662) at

Techniques: