Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Regulation of LPCAT3 mRNA expression by PPARδ activation. A, HepG2 cells were seeded in a 12-well cell culture plate. After 24 h, cells were transduced with Ad-shLacZ (50 MOI) or Ad-shPPARδ (50 MOI) adenovirus in duplicate wells per transduction. Fresh cell culture medium was replaced 5 h later, and cells were incubated for a further 48 h. Total RNA was collected from transduced cells for quantitative PCR analysis. Bars, mean ± S.E. (error bars) of two RNA samples with triplicate measurement per RNA sample. **, p < 0.01; ***, p < 0.001. The data shown are representative of two independent assays. B, HepG2 cells were transduced with Ad-GFP (50 MOI) or Ad-PPARδ (50 MOI) adenoviruses in duplicate wells per transduction. Fresh cell culture medium was replaced 5 h later, and cells were incubated for a further 24 h and then subsequently treated with 1 μm GW0742 or DMSO. Total RNA was collected from transduced cells for quantitative PCR analysis. Bars, mean ± S.E. of two RNA samples with triplicate measurement per RNA sample. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The data shown are representative of two independent assays. C, real-time PCR quantification of LPCAT3 mRNA expression from HepG2, Huh7, and Hepa 1-6 cells treated with GW0742 (1 μm), L165041 (20 μm), or DMSO for 24 h. LPCAT3 expression levels were normalized to GAPDH mRNA levels, where the relative expression of LPCAT3 mRNA in DMSO-treated cells was set at 1. D and E, qRT-PCR quantification of LPCAT3 or ACSL4 mRNA expression from HepG2 cells treated with L165041 at the indicated concentrations. Bars, mean ± S.E. of triplicate measurements of each RNA sample. The data shown are representative of two independent assays. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with DMSO-treated samples. F, C57BL/6J mice fed a normal chow diet were injected with Ad-sh-mPPARδ (n = 4) or Ad-shLacZ (n = 4). Ten days after injection, mice were sacrificed for liver tissue collection. qRT-PCR was used to determine the relative expression levels (mean ± S.E., n = 4/group) of individual mRNAs after normalization with GAPDH mRNA levels. *, p < 0.05; **, p < 0.001, compared with the vehicle group, which was set at 1.

Article Snippet: Constructions of Ad-shLPCAT3 Adenoviral Vector for LPCAT3 Knockdown in Mouse Liver An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Expressing, Activation Assay, Cell Culture, Transduction, Incubation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Injection

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Up-regulation of human LPCAT3 promoter activity in HepG2 cells by PPARδ agonists and LXR agonist. A, diagrammatic representation of human LPCAT3 promoter luciferase reporter construct. B, relative luciferase activities from HepG2 cells transfected with pLPCAT3-Luc promoter luciferase plasmid or pGL3-basic vector. Data represent summarized results (mean ± S.E. (error bars)) of 4–6 replicates/treatment and are expressed as ratio of luciferase/β-gal activity from each sample, where the relative luminescence from cells transfected with promoterless pGL3-basic vector and treated with DMSO is set to 1. *, p < 0.05 compared with DMSO-treated samples. The data shown are representative of three separate transfection experiments.

Article Snippet: Constructions of Ad-shLPCAT3 Adenoviral Vector for LPCAT3 Knockdown in Mouse Liver An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Activity Assay, Luciferase, Construct, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Mapping the functional PPRE site in human LPCAT3 promoter. A, diagrammatic representation of human LPCAT3 promoter luciferase reporter constructs of wild type and PPRE mutants. B, relative luciferase activities from control and L165041-treated Huh7 cells transfected with LPCAT3 promoter wild type and PPRE site-mutated reporter constructs or pGL3-basic vector. Data represent summarized results (mean ± S.E.) of 4 replicates/treatment and are expressed as the ratio of luciferase/β-gal activity from each sample. ***, p < 0.001 compared with DMSO-treated samples. C, after transfection, cells were treated with DMSO as control, 1 μm GW3965, 10 μm L165041, or GW3965 + L165041 for 24 h before cell lysis for the luciferase and β-gal activity assay. The graph represents relative luciferase activities from control and treated HepG2 cells transfected with LPCAT3 promoter wild type and PPRE site-mutated reporter constructs or pGL3-basic vector. Data represent summarized results (mean ± S.E. (error bars)) of 4 replicates/treatment and are expressed as the ratio of luciferase/β-gal activity from each sample. *, p < 0.05; ***, p < 0.001 compared with DMSO-treated samples. The data shown are representative of two separate transfection experiments.

Article Snippet: Constructions of Ad-shLPCAT3 Adenoviral Vector for LPCAT3 Knockdown in Mouse Liver An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Functional Assay, Luciferase, Construct, Control, Transfection, Plasmid Preparation, Activity Assay, Lysis

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: EMSA and ChIP analyses of PPARδ association with PPRE1 site of LPCAT3 promoter in vitro and in vivo. A, chemiluminescent signal from cross-linked nylon membrane, where biotin-5′-end-labeled LPCAT3-PPRE1 (lanes 1–4) was incubated with 200 ng of PPARδ and 100 ng of RXRα recombinant proteins in the absence (lane 2) or presence of a 100-fold molar excess of unlabeled wild type probe (lane 3) or mutated probe (lane 4). B, ChIP analysis on HepG2 cells treated with DMSO or L165041 (10 and 20 μm), where rabbit anti-PPARδ or isotype IgG antibody-immunoprecipitated DNA samples and input DNA samples were PCR-amplified with primers specific for the LPCAT3-PPRE1 and LPCAT3-PPRE2 promoter region. The PCR products were separated on a 2% agarose gel and stained with ethidium bromide. C, real-time quantitative PCR was performed using the two primer sets and chromatin DNA contained in the immunoprecipitates. Values from IgG-immunoprecipitated samples were arbitrarily set to 1.

Article Snippet: Constructions of Ad-shLPCAT3 Adenoviral Vector for LPCAT3 Knockdown in Mouse Liver An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: In Vitro, In Vivo, Membrane, Labeling, Incubation, Recombinant, Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Staining, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Administering L165041 to mice increases hepatic Lpcat3 mRNA and LPCAT enzyme activity along with elevations of Acsl4 and other PPARδ-target genes. Male C57BL/6 mice were gavaged daily with 40 mg/kg L165041 (n = 5) or vehicle (n = 5). After 7 days of treatment, the liver was excised, and the following were measured. A, all isoforms of Lpcat mRNAs. B, D, and F, qRT-PCR was conducted to determine the relative expression levels (mean ± S.E. (error bars), n = 5/group) of individual mRNAs after normalization with GAPDH mRNA levels. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with the vehicle group, which was set at 1. C, total LPCAT activity (mean ± S.E., n = 5/group). ***, p < 0.001, compared with the control group. E, Western blotting analysis of hepatic ACSL4 protein levels.

Article Snippet: Constructions of Ad-shLPCAT3 Adenoviral Vector for LPCAT3 Knockdown in Mouse Liver An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Depletion of hepatic LPCAT3 expression in mice fed a chow diet attenuated PPARδ-mediated activation of hepatic genes in the FA metabolic pathway. Male C57BL/6J mice (n = 5 animals/treatment) were retro-orbitally injected with 3 × 109 IFU/mouse of Ad-shLacZ or Ad-shLPCAT3 adenovirus particles. Three days after injection, mice were orally treated with L165041 (40 mg/kg) or vehicle for 7 days. Four-h-fasted serum samples were collected at the experimental termination, and the liver was excised for the total LPCAT activity assay (A and B) and gene expression analysis (C). Statistical analysis was performed using Student's t test for the enzyme assay (A and B), and one-way ANOVA with Dunnett post hoc test was performed in gene expression analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with the vehicle group injected with Ad-shLacZ. V, vehicle; L, L165041. Error bars, S.E.

Article Snippet: Constructions of Ad-shLPCAT3 Adenoviral Vector for LPCAT3 Knockdown in Mouse Liver An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Expressing, Activation Assay, Injection, Activity Assay, Gene Expression, Enzymatic Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: Effects of hepatic LPCAT3 knockdown on serum and hepatic lipid levels of mice with or without L165041 treatment. Male C57BL/6J mice (5 mice/group) were injected with 3 × 109 IFU/mouse of Ad-shLacZ or Ad-shLPCAT3 adenovirus particles. Three days after injection, mice were orally treated with L165041 (40 mg/kg) or vehicle for 7 days. Four-h-fasted serum samples were collected at the experimental termination, and the liver was excised. Serum TG (A), TC (B), PL (C), and NEFA (D) were measured. Liver lipids were extracted and used to measure hepatic TG (E), TC (F), PL (G), and NEFA (H). Statistical analysis was performed using one-way ANOVA with Dunnett's post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with the vehicle group injected with Ad-shLacZ. V, vehicle; L, L165041. Error bars, S.E.

Article Snippet: Constructions of Ad-shLPCAT3 Adenoviral Vector for LPCAT3 Knockdown in Mouse Liver An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Knockdown, Injection

Journal: The Journal of Biological Chemistry

Article Title: Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ *

doi: 10.1074/jbc.M116.743575

Figure Lengend Snippet: qRT-PCR primer, EMSA probes, promoter cloning primers, and ChIP primer sequences

Article Snippet: Constructions of Ad-shLPCAT3 Adenoviral Vector for LPCAT3 Knockdown in Mouse Liver An adenovirus (Ad-shLPCAT3) expressing an shRNA targeting mouse LPCAT3 mRNA sequence (5′-GCCAATCTACTACGATTGTAT-3′) was generated and amplified by Vector Biolabs (Malvern, PA) for in vivo knockdown of LPCAT3 expression in mouse liver.

Techniques: Cloning

Journal: Journal of acquired immune deficiency syndromes (1999)

Article Title: Brief Report: Rebound HIV viremia with meningoencephalitis following antiretroviral therapy interruption after allogeneic bone marrow transplant

doi: 10.1097/QAI.0000000000002862

Figure Lengend Snippet: Phylogenetic reconstruction of replication-competent viral outgrowth 25 and 100 days pre-alloBMT with corresponding HIV-1 DNA and cell-associated RNA from PBMCs and rCD4+ T-cells. In addition, rebound viremia from plasma and CSF 156-days post-alloBMT following self-interruption of ART are included. The sequences that had the M184I mutation are labeled. Shaded regions indicate predicted APOBEC hypermutated sequences using HyperMut (p<0.05). Colors indicate sample isolate and shape for time point. The scale bar is set to 5 nucleotides.

Article Snippet: HIV plasma-RNA was 25,518 copies/mL and HIV CSF-RNA was 17,000 copies/mL (Abbott Laboratories, IL).

Techniques: Mutagenesis, Labeling

Journal: British Journal of Clinical Pharmacology

Article Title: Therapeutic nucleic acids: current clinical status

doi: 10.1111/bcp.12987

Figure Lengend Snippet: Registered clinical studies with siRNA

Article Snippet: {"type":"clinical-trial","attrs":{"text":"NCT02110563","term_id":"NCT02110563"}} NCT02110563 ; 2014; United States of America , Dicerna Pharmaceuticals, Inc. , Multiple Myeloma Non‐Hodgkins Lymphoma , Phase 1; MYC oncoprotein.

Techniques: Transplantation Assay

Journal: Blood Cancer Journal

Article Title: PICH deficiency limits the progression of MYC-induced B-cell lymphoma

doi: 10.1038/s41408-024-00979-y

Figure Lengend Snippet: A Representative immunoblots of PICH protein levels in Ramos (left) and Raji (right) BL cells seven days after transduction with lentiviruses expressing the indicated shPICH or shControl (shCTRL). β-Actin was used as loading control. B Cell proliferation curve of Ramos (left) and Raji BL cells (right) infected with shCTRL or the indicated shPICH. PICH KD cells proliferated at a lower rate than shCTRL cells and stopped proliferating after four passages. Data shown correspond to a representative experiment (with technical triplicates) out of three biological replicates. The corresponding doubling times (DT) were calculated by non-linear regression and are shown in each graph. SEMs from each data point are indicated and the p -value for each dataset is shown. ** p < 0.01; **** p < 0.0001. C Representative images of Ramos (top) and Raji (bottom) BL cells 10 days after infection with either shCTRL or shPICH. The images were captured at 4x magnification with a light optical microscope. Scale bar, 50 μm. Images were acquired immediately prior to the addition of MTT into the wells. D Relative cell viability of Ramos (left) and Raji (right) BL cells upon depletion of PICH compared to transfection with control shRNA (shCTRL) assessed by MTT assay 10 days after infection. Data are represented as relative absorbance at 570 nm and correspond to four independent experiments with six technical replicates for each experiment. Mean and SEMs are indicated. Significance was assessed by unpaired t -test. **** p ≤ 0.0001. E Relative cell viability of Ramos (left) and Raji (right) BL cells upon depletion of PICH compared to transfection with control shRNA (shCTRL) determined by cell counting after labeling cells with trypan blue 10 days after infection. Histograms show the mean of the number of non-trypan blue-stained live cells of PICH depleted cells relative to shCTRL cells and correspond to three independent experiments with three technical replicates for each experiment. Mean and SEMs are indicated. Significance was assessed by unpaired t -test. *** p ≤ 0.001; **** p ≤ 0.0001. F Cell viability of Ramos (left) and Raji (right) BL cells 10 days after transduction with lentiviruses expressing the indicated shPICH or shCTRL. Growing cells were incubated with Hoechst 33342 and To-Pro-3 dyes for 30 min. After labeling, images were acquired with a High-Content Screening System (MetaXpress) at 10× magnification. Quantification of Hoechst 33342-positive and To-Pro-3 positive cells were performed with the MetaXpress High-Content Image Analysis Software. Live cells were calculated by subtracting the number of To-Pro-3 positive cells from the Hoechst 33342-positive total cell number. Data correspond to three independent experiments with three technical replicates for each experiment. Mean and SEMs are indicated. Significance was assessed by unpaired t -test. **** p ≤ 0.0001. G Effect of PICH KD on the apoptosis of Ramos and Raji BL cancer cells measured by Annexin V-FITC/Propidium iodide and flow cytometry 7 days after infection with either shCTRL or shPICH. The percentage of apoptotic (Annexin V-positive) cells is indicated. Mean and SEMs are indicated. Significance was assessed by unpaired t -test. * p ≤ 0.05; ** p ≤ 0.01.

Article Snippet: HEK293T cells were used for the synthesis of third-generation lentiviruses containing short hairpin RNA (shRNA) targeting human PICH (shPICH) or the shRNA control pLKO.1 (shCtrl) (#8453, Addgene) vector.

Techniques: Western Blot, Transduction, Expressing, Control, Infection, Microscopy, Transfection, shRNA, MTT Assay, Cell Counting, Labeling, Staining, Incubation, High Content Screening, Software, Flow Cytometry