Journal: Scientific Reports

Article Title: Transcriptomic sex differences in sensory neuronal populations of mice

doi: 10.1038/s41598-020-72285-z

Figure Lengend Snippet: Highly expressing and/or regulated sex-dependent DEGs in sensory neurons.

Article Snippet: Specific primers were Mm00658576_m1 for Cd74, Mm01700423_m1 for Islr, Mm01191753_m1 for Bgn, Mm00470429_g1 for Mxra8, Mm00435846_m1 for Pkib and Mm00547130_s1 for Arxes2.

Techniques: Expressing, Functional Assay

Journal: Journal of Lipid Research

Article Title: Characterization of a cholesterol response element (CRE) in the promoter of the cholesteryl ester transfer protein gene: functional role of the transcription factors SREBP-1a, -2, and YY1.

doi: 10.1016/s0022-2275(20)33490-8

Figure Lengend Snippet: Fig. 1. CETP mRNA levels in confluent SW872 cells are modu- lated by lipids. The effect of lipids and 25-OH cholesterol on levels of CETP mRNA was determined by incubating SW872 cells for 24 h in DMEM/HAM F12 (3:1) supplemented with either lipoprotein- depleted serum (LPDS) or complete serum (CS) in the presence or absence of 5 mm 25-OH cholesterol. Cells were harvested and poly(A)1 RNA was extracted using the polyATtract mRNA isolation system. (A) Four mg of poly(A)1 RNA was size-fractionated on a agarose/formaldehyde gel and transferred to a nylon membrane. CETP mRNA was detected by overnight hybridization at 428C against an [a32P]dCTP random-primed cDNA corresponding to the cod- ing region of this gene (specific activity of 5–8 3 108 cpm/mg). To correct for differences in RNA loading, blots were also hybridized with a b-actin cDNA probe. 1; LPDS, 2; CS, 3; 25-hydroxy-choles- terol. (B) Graphic representation of three Northern blot analyses. Results are expressed as the percentage of treated samples as com- pared to the LPDS sample; P , 0.05 for CS and 25-OH as compared to LPDS.

Article Snippet: The human liposarcoma cell line SW872 (American Type Culture Collection, Rockville, MD) was cultured in Dulbecco’s modified Eagle’s/HAM F12 (3:1) (Life Technologies, Burlington, ON) supplemented with 5% fetal bovine serum (Life Technologies, Burlington, ON), 10 m m HEPES, and 50 m g/ml gentamycin (NovoPharm, Toronto, ON) at 37 8 C in the presence of 5% CO 2 .

Techniques: Isolation, Membrane, Hybridization, Random Primed, Activity Assay, Northern Blot

Journal: Journal of Lipid Research

Article Title: Characterization of a cholesterol response element (CRE) in the promoter of the cholesteryl ester transfer protein gene: functional role of the transcription factors SREBP-1a, -2, and YY1.

doi: 10.1016/s0022-2275(20)33490-8

Figure Lengend Snippet: Fig. 2. Deletion of the region spanning nucleotides 2361 to 2138 of the human CETP promoter results in the lost of lipid-mediated regulation of the gene. (A) Serial deletion derivatives of the CETP gene 59 flanking region were subcloned into the pGL3-Basic luciferase reporter vector. The effect of lipids on the expression of each construct was then assessed by transfecting SW872 cells with 3 mg of either CETP600LUC, CETP361LUC, CETP125LUC, or pGL3-Basic and 0.5 mg pRL-TK using the calcium phosphate–DNA precipitate method formed in BES. Cellular protein extracts were isolated 24 h after transfection and luciferase activities for both the Renilla and firefly gene were determined using the Dual-Luciferase™ reporter assay system. The luciferase activity of each construct was then normalized to the Renilla activity. Results are expressed as fold induction as compared to the LPDS-treated sample of each construct. All experiments were performed in triplicate on two separate occasions; * CETP361LUC, P , 0.05 for CS and 25-OH as compared to LPDS; # CETP600LUC P , 0.05 for CS and 25-OH as compared to LPDS. (B) Five mg of the luciferase construct CETP361LUC or pGL3-Basic were transfected into SW872 cells which were subsequently maintained in LPDS media with in- creasing concentrations of HDL particle.

Article Snippet: The human liposarcoma cell line SW872 (American Type Culture Collection, Rockville, MD) was cultured in Dulbecco’s modified Eagle’s/HAM F12 (3:1) (Life Technologies, Burlington, ON) supplemented with 5% fetal bovine serum (Life Technologies, Burlington, ON), 10 m m HEPES, and 50 m g/ml gentamycin (NovoPharm, Toronto, ON) at 37 8 C in the presence of 5% CO 2 .

Techniques: Luciferase, Plasmid Preparation, Expressing, Construct, Isolation, Transfection, Reporter Assay, Activity Assay

Journal: Journal of Lipid Research

Article Title: Characterization of a cholesterol response element (CRE) in the promoter of the cholesteryl ester transfer protein gene: functional role of the transcription factors SREBP-1a, -2, and YY1.

doi: 10.1016/s0022-2275(20)33490-8

Figure Lengend Snippet: Fig. 3. (A) Schematic representation of the 2361 to 2138 region of the CETP promoter depicting the architecture of the cholesterol response element (CRE) and surrounding sequences. The se- quence of the human CETP CRE (HumCETP) is shown and com- pared to the SRE of the hamster HMG-CoA reductase gene (Ham- HMG). The element contains two direct repeats (indicated in bold) which have been denoted as cre-1 and cre-2 (arrows). Similar sequences in the SRE of the hamster HMG-CoA reductase gene (depicted in italics) were shown to interact with the transcription factors YY1 and SREBP-1a. This region also contains a C/EBP site, which is important for hepatic expression of the CETP gene. (B) Gel mobility shift assays were performed using a labeled oligonucle- otide (10,000 cpm/reaction) corresponding to the CRE (2213 to 2186) of the CETP gene as shown in Fig. 3A and nuclear protein extracts (4 mg) isolated from SW872 cells grown in CS or LPDS. NP depicts no protein control.

Article Snippet: The human liposarcoma cell line SW872 (American Type Culture Collection, Rockville, MD) was cultured in Dulbecco’s modified Eagle’s/HAM F12 (3:1) (Life Technologies, Burlington, ON) supplemented with 5% fetal bovine serum (Life Technologies, Burlington, ON), 10 m m HEPES, and 50 m g/ml gentamycin (NovoPharm, Toronto, ON) at 37 8 C in the presence of 5% CO 2 .

Techniques: Expressing, Mobility Shift, Labeling, Isolation, Control

Journal: Journal of Lipid Research

Article Title: Characterization of a cholesterol response element (CRE) in the promoter of the cholesteryl ester transfer protein gene: functional role of the transcription factors SREBP-1a, -2, and YY1.

doi: 10.1016/s0022-2275(20)33490-8

Figure Lengend Snippet: Fig. 5. SREBP-1a but not YY1 trans-activates only the luciferase activity of the construct harboring the CRE of CETP. Transient co-transfection studies using SW872 cells were performed with 3 mg of either luciferase reporter construct CETP361LUC or CETP125LUC and increasing amounts of expression vectors for: (A) YY1 or (B) SREBP-1a or (C) SREBP-2. As control, 3 mg of pGL3-basic was also transfected with increasing amounts of YY1, SREBP-1a, and -2. The total amount of DNA transfected was maintained at 5 mg/transfec- tion using herring testis DNA. Firefly luciferase activity was assayed as described in Fig. 2 and normalized to Renilla luciferase activity (YY1) or total cellular protein content (SREBP-1a and -2). Results are expressed as fold induction compared to control sample. Experiments were performed in triplicate.

Article Snippet: The human liposarcoma cell line SW872 (American Type Culture Collection, Rockville, MD) was cultured in Dulbecco’s modified Eagle’s/HAM F12 (3:1) (Life Technologies, Burlington, ON) supplemented with 5% fetal bovine serum (Life Technologies, Burlington, ON), 10 m m HEPES, and 50 m g/ml gentamycin (NovoPharm, Toronto, ON) at 37 8 C in the presence of 5% CO 2 .

Techniques: Luciferase, Activity Assay, Construct, Cotransfection, Expressing, Control, Transfection

Journal: Journal of Lipid Research

Article Title: Characterization of a cholesterol response element (CRE) in the promoter of the cholesteryl ester transfer protein gene: functional role of the transcription factors SREBP-1a, -2, and YY1.

doi: 10.1016/s0022-2275(20)33490-8

Figure Lengend Snippet: Fig. 6. Transcription factors YY1 and SREBP-1a act together to promote CETP300LUC luciferase activity. Transient co-transfection studies using SW872 cells were performed with 3 mg of the luciferase reporter construct CETP361LUC, 500 ng of YY1 expression vector, and increasing amounts of the expression vector for SREBP-1a. The total amount of DNA transfected was maintained at 5 mg/transfec- tion using herring testis DNA. Firefly luciferase activity was assayed as described in Fig. 2 and normalized to total cellular protein con- tent. Results are expressed as fold induction compared to control sample.

Article Snippet: The human liposarcoma cell line SW872 (American Type Culture Collection, Rockville, MD) was cultured in Dulbecco’s modified Eagle’s/HAM F12 (3:1) (Life Technologies, Burlington, ON) supplemented with 5% fetal bovine serum (Life Technologies, Burlington, ON), 10 m m HEPES, and 50 m g/ml gentamycin (NovoPharm, Toronto, ON) at 37 8 C in the presence of 5% CO 2 .

Techniques: Luciferase, Activity Assay, Cotransfection, Construct, Expressing, Plasmid Preparation, Transfection, Control

Journal: Journal of Lipid Research

Article Title: Characterization of a cholesterol response element (CRE) in the promoter of the cholesteryl ester transfer protein gene: functional role of the transcription factors SREBP-1a, -2, and YY1.

doi: 10.1016/s0022-2275(20)33490-8

Figure Lengend Snippet: Fig. 7. The CRE is the functional cis-acting element involved in lipid-mediated up-regulation of CETP gene expression through its interaction with SREBP-1a. (A) Mutagenesis of the CRE was per- formed by PCR-mediated site directed mutagenesis. The mutated nu- cleotides were selected due to the critical role that each plays in bind- ing SREBP-1a and YY1 to the SRE of the HMG-CoA reductase gene. Compared to a wild-type plasmid wtCETP300LUC, the mutated plas- mid, mutCETP300LUC, demonstrated a significant decrease in the ability of SREBP-1a to induce luciferase activity (2- fold vs. 4-fold in- crease). Three copies of the CRE were also concatemerized and cloned into the pGL3-promoter vector to produce pGL3-PCRE3. The ability of these constructs to respond to SREBP-1a (800 ng) was com- pared to parental vectors (wtCETP300LUC and pGL3-Promoter). Three mg of either wtCETP300LUC (Wt), mutCETP300LUC (Mut), pGL3-Promoter (pGL3-P), or pGL3-PCRE3 were co-transfected along with 800 ng of SREBP-1a into SW872 cells. Firefly luciferase activity was measured as described in Fig. 2 and normalized to total cellu- lar protein content. Results are expressed as fold induction of basal luciferase activity obtained in the absence of SREBP-1a. Although 800 ng of SREBP-1a resulted in a 2.7-fold increase in the luciferase activity of pGL3-promoter, the insertion of three copies of the CRE into the vector resulted in a further 10-fold increase in activ-

Article Snippet: The human liposarcoma cell line SW872 (American Type Culture Collection, Rockville, MD) was cultured in Dulbecco’s modified Eagle’s/HAM F12 (3:1) (Life Technologies, Burlington, ON) supplemented with 5% fetal bovine serum (Life Technologies, Burlington, ON), 10 m m HEPES, and 50 m g/ml gentamycin (NovoPharm, Toronto, ON) at 37 8 C in the presence of 5% CO 2 .

Techniques: Functional Assay, Gene Expression, Mutagenesis, Plasmid Preparation, Luciferase, Activity Assay, Clone Assay, Construct, Transfection