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: PloS one

Article Title: Bone marrow deficiency of MCPIP1 results in severe multi-organ inflammation but diminishes atherogenesis in hyperlipidemic mice.

doi: 10.1371/journal.pone.0080089

Figure Lengend Snippet: Figure 9. MCPIP1 deficiency increased macrophage cholesterol efflux. A. Bone marrow-derived macrophages were loaded with ac-LDL and cholesterol efflux to DMEM, apoAI and HDL was measured. Data were presented as mean 6 SEM. N = 3 in each group. B. Bone marrow-derived macrophages were cultured in DMEM for 48 h with or without ac-LDL. Cell lysates were used for western blotting analysis for ABCA1 and ABCG1. Representative western blotting result was shown. C. Quantitative analysis of the western blots. Data were presented as mean 6 SEM. N = 4 in each group. Open column: WT macrophages; Black column, MCPIP12/2 macrophages. doi:10.1371/journal.pone.0080089.g009

Article Snippet: Rabbit antimouse ABCA1 and anti-mouse ABCG1 antibodies were from Novus Biologicals (Littleton, CO).

Techniques: Derivative Assay, Cell Culture, Western Blot

Journal: Journal of Lipid Research

Article Title: HBV-miR-3 induces hepatic cholesterol accumulation by targeting ABCA1 : Evidence for potential benefits of statin usage

doi: 10.1016/j.jlr.2025.100866

Figure Lengend Snippet: HBV-miR-3 downregulates h ABCA1 expression. A: Upper panel: Venn diagram showing common genes between two data sets (a) downregulated DEGs (microarray data) and (b) computationally predicted host genes with putative HBV-miR-3 binding sites. Lower panel: Computed Mfe of binding between the potential miRNA-mRNA pairs and their respective expression level of the gene observed in the microarray data. B: Data extracted from published GEO Datasets: ABCA1 transcript levels in HBV infected liver biopsies compared with normal liver. C: Schematic representation of predicted HBV-miR-3 binding site in ABCA1 mRNA. D: qRT PCR analysis of ABCA1 mRNA level in cells (Huh7, HepG2) transfected with pHBV-miR-3 compared to pcDNA3.1+ transfected cells. We used 18s rRNA was used as internal control. E: Analysis of luciferase reporter activity of ABCA1-3′UTR reporter constructs having either wildtype or deleted HBV-miR-3 binding sites in the presence or absence of HBV-miR-3 in both (Huh7, HepG2) cell lines. Firefly luciferase counts were normalized with Renilla luciferase counts. The bar graphs represent mean (±SD) of three independent experiments. F: Left panel: Immunostaining for ABCA1 protein in cells (Huh7, HepG2) transfected with pHBV-miR-3 compared to pcDNA3.1+ transfected cells. The cells were counterstained with nuclear stain DAPI. Right Panel: Fluorescence intensity was quantified in each micrograph and expressed as the mean ± standard errors (n = 7) in arbitrary units (a.u). Scale bar: 100 μM. ∗ P ≤ 0.05.

Article Snippet: For immunofluorescence staining, ABCA1 Antibody (1276B) [NBP2-54792, Novus Biologicals] was used at a 1:100 dilution overnight, and Alexa Fluor 555 goat anti-rabbit IgG [A-11008, Thermo Fisher Scientific] at a 1:800 dilution for 2 h was used as a secondary antibody.

Techniques: Expressing, Microarray, Binding Assay, Infection, Quantitative RT-PCR, Transfection, Control, Luciferase, Activity Assay, Construct, Immunostaining, Staining, Fluorescence

Journal: Journal of Lipid Research

Article Title: HBV-miR-3 induces hepatic cholesterol accumulation by targeting ABCA1 : Evidence for potential benefits of statin usage

doi: 10.1016/j.jlr.2025.100866

Figure Lengend Snippet: Correlation analysis intrahepatic HBV-miR-3 expression levels in liver biopsies of HBV patients (N = 20) with other parameters. A: Scatter plot demonstrating the correlation (r s : Spearman rank correlation coefficient) between intrahepatic HBV-miR-3 levels and intrahepatic HBV DNA (HBV load) in chronic HBV patients (n = 20). B: A plot showing the range of intrahepatic HBV-miR-3 levels in chronic HBV patients, wherein biopsies with HBV-miR-3 copies ≥ 100 copies/100ngRNA are grouped as patients with high HBV-miR-3 and those with HBV-miR-3 copies ≤ 100 copies/100ngRNA (lower than the linear range) are grouped as patients with low HBV-miR-3. (# HBV-miR-3 levels in the biopsies were normalized HBV-miR-3/18srRNA/100 ng RNA). C: Violin plots demonstrating the distribution of intrahepatic viral (HBV) load in biopsies with high HBV-miR-3 expression compared to biopsies with low HBV-miR-3 expression. D: Violin plots showing the distribution of ABCA1 levels in biopsies with high HBV-miR-3 expression compared to those with low HBV-miR-3 expression. E: Frequency of hepatic steatosis in biopsies with high HBV-miR-3 expression and low HBV-miR-3 expression. F: Frequency of liver cirrhosis in biopsies with high HBV-miR-3 expression and low HBV-miR-3 expression. ∗ P ≤ 0.05, n.s.: non-significant.

Article Snippet: For immunofluorescence staining, ABCA1 Antibody (1276B) [NBP2-54792, Novus Biologicals] was used at a 1:100 dilution overnight, and Alexa Fluor 555 goat anti-rabbit IgG [A-11008, Thermo Fisher Scientific] at a 1:800 dilution for 2 h was used as a secondary antibody.

Techniques: Expressing