Journal: International Journal of Biological Sciences

Article Title: Mechanism of AMPK Suppression of LXR-dependent Srebp-1c Transcription

doi:

Figure Lengend Snippet: AMPK activation suppresses LXR ligand-mediated induction of Srebp-1c promoter activity in McA-RH7777 cells. A , Diagram of plasmids containing the wild-type or mutant mouse Srebp-1c promoter linked to the firefly luciferase reporter gene. B , Luciferase reporter assay. On Day 0, McA-RH7777 cells were plated at density of 5 x 10 4 cells per well in 24-well plates in Medium A supplemented with 10% FBS and incubated at 37°C in a 5% CO 2 incubator. On Day 1, cells were washed with 0.5 ml PBS and 0.5 ml fresh Medium A was added to each well before transfection. Cells were cotransfected with plasmid D and a control plasmid as described in Materials and Methods . Six hours after transfection, cells were washed with 0.5 ml PBS, switched to Medium A supplemented with 10% delipidated FBS and 0.5 mM AICAR or metformin or various amounts of T0901317, and incubated for 16 hours at 37°C and 5% CO 2 . On Day 2, cells were washed with 0.5 ml PBS, and luciferase and beta-gal activities were measured as described in the Materials and Methods . Normalized firefly luciferase activity without treatment was arbitrarily set as 1.

Article Snippet: On Day 2, cells were washed with 0.5 ml PBS and firefly luciferase activities were measured using the Luciferase Reporter Assay System and Turner Designs TD-20/20 Luminometer.

Techniques: Activation Assay, Activity Assay, Mutagenesis, Luciferase, Reporter Assay, Incubation, Transfection, Plasmid Preparation, Control

Journal: International Journal of Biological Sciences

Article Title: Mechanism of AMPK Suppression of LXR-dependent Srebp-1c Transcription

doi:

Figure Lengend Snippet: Activation of AMPK decreases the fold induction of wild-type and mutant Srebp-1c promoter activities by LXR ligand T0901317 in McA-RH7777 cells treated with compactin. McA-RH7777 cells were cultured, transfected, and treated with AICAR (0.5 mM) or metformin (0.5 mM) with or without T0901317 (1 μM) in the presence of compactin (50 μM compactin + 50 μM sodium mevalonate) as in Fig. . Cells were lysed and luciferase reporter assays were performed as in Fig. with ( A ) wild-type Srebp-1c promoter construct pD, ( B ) pM31 (-LXRE1, 2), ( C ) pM24 (-SRE), and ( D ) pM34 (-LXRE1, 2/SRE). The value on each column represents the fold change (the average of three assays) as compared with control (-AICAR/Metformin/T0901317). White bar, - T0901317; grey bar, + T0901317. *, P<0.05 as compared with control (+ T0901317) without AICAR/metformin treatment for each promoter construct.

Article Snippet: On Day 2, cells were washed with 0.5 ml PBS and firefly luciferase activities were measured using the Luciferase Reporter Assay System and Turner Designs TD-20/20 Luminometer.

Techniques: Activation Assay, Mutagenesis, Cell Culture, Transfection, Luciferase, Construct, Control

Journal: Virus Research

Article Title: Porcine reproductive and respiratory syndrome virus (PRRSV) could be sensed by professional beta interferon-producing system and had mechanisms to inhibit this action in MARC-145 cells

doi: 10.1016/j.virusres.2010.07.028

Figure Lengend Snippet: PRRSV phosphorylated IRF-3 and weakly activated the IFN-β promoter in MARC-145 at 24 h p.i., but their activities were rapidly inhibited in the following infection. (A) MARC-145 cells were infected with PRRSV at an MOI of 0.05. After 6 h, 18 h, 30 h respectively, the cells were co-transfected with p-284 Luc and phRL-TK, and 18 h later, cells were harvested for dual luciferase reporter assay system in MicroBeta-1450. Cells were transfected with Poly(I:C) for 9 h as positive controls. (B) MARC-145 cells were infected with PRRSV at an MOI of 0.05, and harvested at 24, 36, 48 h p.i. for the immunoblots of IRF-3 and pIRF-3. The results shown were from one of three independent experiments with similar observations, and each including three or two replicates. Control (Con): Sham virus mentioned in part of Section were added into MARC-145 cells. *: p < 0.05 compared to the control.

Article Snippet: The firefly- luciferase plasmid for monitoring IFN-β promoter activation (p-284 Luc) was cloned from genetic DNA of MARC-145 cells and was ligated into pGL-417 (Prpmega). p55C1B Luc ( , , ), a firefly- luciferase reporter gene plasmid containing repetitive pIRF-3 binding sites, was kindly provided by Dr. Takashi Fujita. phRL-TK (Promega), contained a Renilla -luciferase reporter gene, was used as an internal control in dual luciferase reporter assay system. pcDNA3.1-His was constructed by using His primers.

Techniques: Infection, Transfection, Luciferase, Reporter Assay, Western Blot, Control, Virus

Journal: Virus Research

Article Title: Porcine reproductive and respiratory syndrome virus (PRRSV) could be sensed by professional beta interferon-producing system and had mechanisms to inhibit this action in MARC-145 cells

doi: 10.1016/j.virusres.2010.07.028

Figure Lengend Snippet: PRRSV nsp1 inhibited the activations of IFN-β promoter and pIRF-3-dependent synthetic promoter (p55C1B Luc) induced by Poly(I:C). (A) The mRNA of nsp1 and N in MARC-145 cells transfected with pcDNA3.1-nsp1 (nsp1), pcDNA3.1-N (N) or pcDNA3.1-His (Con) were analyzed by RT-PCR. Lanes 1–3 were amplified by PRRSV N primers, while lanes 4–6 were amplified by PRRSV nsp1 primers. (B) Western blot analyzed nsp1 and N protein by anti-His primary antibody in MARC-145 transfected with pcDNA3.1-nsp1 (nsp1) or pcDNA3.1-N (N). The control cells were transfected with pcDNA 3.1-His (Con). Since the nsp1 should be cleaved into nsp1α and nsp1β subunits, the arrow-pointed band was only the nsp1β subunits. (C) MARC-145 cells were co-transfected with p-284 Luc, phRL-TK and different expression plasmid, and 24 h later, cells were either mock-treated (Con) or transfected with Poly(I:C) for 6 h. Cells were analyzed by dual luciferase reporter assay. (D) MARC-145 cells were co-transfected with p55C1B Luc, phRL-TK and different expression plasmid. Twenty four hours later, cells were either mock-treated (Con) or transfected with Poly(I:C) for 6 h. Cells were analyzed by dual luciferase reporter assay. LCPS was the luminescence counts per second detected by luminescence counting on the Microbeta-1450. Bars showed relative luciferase activity (Relative LCPS) normalized to phRL-TK activity (relative LCPS = LCPS of firefly- luciferase/LCPS of Renilla- luciferase). Data represented means of three replicates, and experiments were repeated three times. Vector (pcDNA3.1-His) showed in figure was the control plasmid. *: p < 0.05 compared to the cells transfected with pcDNA3.1-His (Con).

Article Snippet: The firefly- luciferase plasmid for monitoring IFN-β promoter activation (p-284 Luc) was cloned from genetic DNA of MARC-145 cells and was ligated into pGL-417 (Prpmega). p55C1B Luc ( , , ), a firefly- luciferase reporter gene plasmid containing repetitive pIRF-3 binding sites, was kindly provided by Dr. Takashi Fujita. phRL-TK (Promega), contained a Renilla -luciferase reporter gene, was used as an internal control in dual luciferase reporter assay system. pcDNA3.1-His was constructed by using His primers.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Control, Expressing, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay

Journal: Virus Research

Article Title: Porcine reproductive and respiratory syndrome virus (PRRSV) could be sensed by professional beta interferon-producing system and had mechanisms to inhibit this action in MARC-145 cells

doi: 10.1016/j.virusres.2010.07.028

Figure Lengend Snippet: PRRSV nsp1 inhibited activities of IFN-β promoter and pIRF-3 dependent promoters (p55C1B Luc) induced by RIG-N, VISA, TRIF or TBK1, but did not inhibit the activities of IFN-β promoter and pIRF-3 dependent promoter induced by IRF-3(5D). MARC-145 cells were co-transfected with p-284 Luc (A) or p55C1B Luc (B), phRL-TK and different expression plasmids. Thirty hours later, cells were harvested for dual luciferase reporter assay. Con: cells transfected with pcDNA3.1-His. Data represented means of three replicates, and experiments were repeated three times. *: p < 0.05 compared to cells transfected with pcDNA3.1-His (Con).

Article Snippet: The firefly- luciferase plasmid for monitoring IFN-β promoter activation (p-284 Luc) was cloned from genetic DNA of MARC-145 cells and was ligated into pGL-417 (Prpmega). p55C1B Luc ( , , ), a firefly- luciferase reporter gene plasmid containing repetitive pIRF-3 binding sites, was kindly provided by Dr. Takashi Fujita. phRL-TK (Promega), contained a Renilla -luciferase reporter gene, was used as an internal control in dual luciferase reporter assay system. pcDNA3.1-His was constructed by using His primers.

Techniques: Transfection, Expressing, Luciferase, Reporter Assay