isre-luc in pgl3-basic luciferase reporter vectors  (Promega)

Journal: PLoS Pathogens

Article Title: A novel role of Zebrafish TMEM33 in negative regulation of interferon production by two distinct mechanisms

doi: 10.1371/journal.ppat.1009317

Figure Lengend Snippet: (A and B) Overexpression of TMEM33 suppresses poly I:C/SVCV-induced IFNφ1pro/ISRE-Luc activation and displays a dose-dependent manner. EPC cells were seeded in 24-well plates and transfected the next day with 250 ng IFNφ1pro-Luc (A) or ISRE-Luc (B) and 25 ng pRL-TK, plus pcDNA3.1-TMEM33 (250 ng or 200/400 ng) or pcDNA3.1(+) (control vector). At 24 h post-transfection, cells were untreated (null) or transfected with poly I:C (1 μg/ml) or treated with SVCV (MOI = 1). Luciferase activities were monitored at 24 h after stimulation. The promoter activity is presented as relative light units (RLU) normalized to Renilla luciferase activity. (C-G) Overexpression of TMEM33 inhibits the expression of ifn (C), vig1 (D), isg15-1 (E), irf7 (F), and rig-i (G) induced by poly I:C in EPC cells. EPC cells seeded in 6-well plates overnight were transfected with 2 μg TMEM33-Myc or empty vector and transfected with poly I:C at 24 h post-transfection. At 24 h after stimulation, total RNAs were extracted to examine the mRNA levels of cellular ifn , vig1 , isg15-1 , irf7 , and rig-i . (H) Effects of TMEM33 RNAi on the expression of endogenous TMEM33. EPC cells were seeded in 6-well plates overnight and transfected with 100 nM si-TMEM33#1, si-TMEM33#2, or si-NC (negative control). At 24 h post-transfection, the cells were transfected with poly I:C or treated with SVCV (MOI = 1). At 24 h post-stimulation, total RNAs were extracted to examine the transcriptional levels of TMEM33. (I and J) Effects of TMEM33 RNAi on the poly I:C/SVCV-induced epc ifn and epc vig1 transcription. EPC cells were seeded in 6-well plates and transfected with 100 nM si-NC or si-TMEM33#2. At 24 h post-transfection, cells were untreated or transfected with poly I:C or treated with SVCV for 24 h before qPCR analysis was performed. The relative transcriptional levels were normalized to the transcription of β-actin and represented as fold induction relative to the transcriptional level in the control cells, which was set to 1. Data were expressed as mean ± SEM, n = 3. Asterisks indicate significant differences from control (*, p < 0.05).

Article Snippet: The plasmids containing zebrafish IFNφ1pro-Luc and ISRE-Luc in the pGL3-Basic luciferase reporter vector (Promega) were constructed as described previously [ ].

Techniques: Over Expression, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Negative Control

Journal: PLoS Pathogens

Article Title: A novel role of Zebrafish TMEM33 in negative regulation of interferon production by two distinct mechanisms

doi: 10.1371/journal.ppat.1009317

Figure Lengend Snippet: (A and B) EPC cells were seeded into 24-well plates overnight and co-transfected with MAVS-, MITA-, or TBK1-expressing plasmid and empty vector or TMEM33-Myc (250 ng or 250/500 ng), plus IFNφ1pro-Luc (A and C) or ISRE-Luc (B and D) at the ratio of 1:1:1. pRL-TK was used as a control. At 24 h post-transfection, cells were lysed for luciferase activity detection. Data were expressed as mean ± SEM, n = 3. Asterisks indicate significant differences from control (*, p < 0.05).

Article Snippet: The plasmids containing zebrafish IFNφ1pro-Luc and ISRE-Luc in the pGL3-Basic luciferase reporter vector (Promega) were constructed as described previously [ ].

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

Journal: PLoS Pathogens

Article Title: A novel role of Zebrafish TMEM33 in negative regulation of interferon production by two distinct mechanisms

doi: 10.1371/journal.ppat.1009317

Figure Lengend Snippet: (A and B) TMEM33-ΔTM1 and TMEM33-ΔTM2 have no effect on poly I:C/SVCV-induced IFNφ1pro/ISRE-Luc activation. EPC cells were seeded in 24-well plates and transfected the next day with 250 ng IFNφ1pro-Luc (A) or ISRE-Luc (B) and 25 ng pRL-TK, plus 250 ng TMEM33-Myc, TMEM33-ΔTM1-Myc, TMEM33-ΔTM2-Myc, TMEM33-ΔTM3-Myc, or pCMV-Myc (control vector). At 24 h post-transfection, cells were untreated (null) or transfected with poly I:C (1 μg/ml) or treated with SVCV (MOI = 1). Luciferase activities were monitored at 24 h after stimulation. The promoter activity is presented as relative light units (RLU) normalized to Renilla luciferase activity. (C and D) TMEM33-ΔTM1 and TMEM33-ΔTM2 have no effect on RLRs-induced IFNφ1pro/ISRE-Luc activation. EPC cells were seeded in 24-well plates and transfected the next day with 250 ng IFNφ1pro-Luc (C) or ISRE-Luc (D) and 250 ng MAVS-Myc, MITA-Myc, or TBK1-Myc and 25 ng pRL-TK, plus 250 ng TMEM33-Myc, TMEM33-ΔTM1-Myc, TMEM33-ΔTM2-Myc, TMEM33-ΔTM3-Myc, or pCMV-Myc (control vector). Luciferase activities were monitored at 24 h after transfection. (E-I) TMEM33-ΔTM1 and TMEM33-ΔTM2 have no effect on poly I:C/SVCV-induced the expression of ifn (E), vig1 (F), isg15-1 (G), irf7 (H), and rig-i (I). EPC cells seeded in 6-well plates overnight were transfected with the indicated plasmids (2 μg each) for 24 h. At 24 h post-transfection, cells were untreated (null) or transfected with poly I:C (1 μg/ml) or stimulated with SVCV (MOI = 1) for 24 h. Total RNAs were extracted to examine the mRNA levels of cellular ifn , vig1 , isg15-1 , irf7 , and rig-i . The relative transcriptional levels were normalized to the transcription of β-actin and represented as fold induction relative to the transcriptional level in the control cells, which was set to 1. Data were expressed as mean ± SEM, n = 3. Asterisks indicate significant differences from control (*, p < 0.05).

Article Snippet: The plasmids containing zebrafish IFNφ1pro-Luc and ISRE-Luc in the pGL3-Basic luciferase reporter vector (Promega) were constructed as described previously [ ].

Techniques: Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Expressing

Journal: Fish & Shellfish Immunology

Article Title: Grass carp reovirus VP56 represses interferon production by degrading phosphorylated IRF7

doi: 10.1016/j.fsi.2020.02.004

Figure Lengend Snippet: VP56 inhibits MAVS-mediated IFN1 activation. (A and B) EPC cells were seeded on 24-well plates overnight and co-transfected with MAVS, IRF7, and pcDNA3.1-VP56 or pcDNA3.1 (+) plus IFN1pro (A) or ISRE-Luc (B) at the ratio of 1:1:1 (0.5 μg for each). pRL-TK was used as a control. At 24 h post transfection, cells were collected for detection of luciferase activities. The promoter activity is presented as relative light units normalized to Renilla luciferase activity. Data were expressed as mean ± SEM, n = 3. Asterisks indicate significant differences from control (*, p < 0.05). All experiments were repeated for at least three times with similar result.

Article Snippet: The ISRE-Luc plasmid in the pGL3-basic luciferase reporter vector (Promega) was constructed as described previously [ ].

Techniques: Activation Assay, Transfection, Luciferase, Activity Assay