Structured Review

Promega prl tk vector
Involvement of PPARα on the cyanidin-induced elevation of TRPM6 and CNNM4 promoter activities. TRPM6 promoter ( A ) or CNNM4 promoter ( B ) luciferase vectors were co-transfected with a <t>pRL-TK</t> vector into MCE301 cells. WT and mutant indicate vectors with wild-type and mutant PPRE sequences, respectively. At 24 h after <t>transfection,</t> the cells were incubated with vehicle, 10 μM cyanidin, cyanidin plus 10 μM GW6471, or cyanidin plus 10 μM GW9662 for 6 h. The relative promoter activity was represented relative to the values in vehicle. n = 3–4. ** p
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1) Product Images from "Cyanidin Increases the Expression of Mg2+ Transport Carriers Mediated by the Activation of PPARα in Colonic Epithelial MCE301 Cells"

Article Title: Cyanidin Increases the Expression of Mg2+ Transport Carriers Mediated by the Activation of PPARα in Colonic Epithelial MCE301 Cells

Journal: Nutrients

doi: 10.3390/nu11030641

Involvement of PPARα on the cyanidin-induced elevation of TRPM6 and CNNM4 promoter activities. TRPM6 promoter ( A ) or CNNM4 promoter ( B ) luciferase vectors were co-transfected with a pRL-TK vector into MCE301 cells. WT and mutant indicate vectors with wild-type and mutant PPRE sequences, respectively. At 24 h after transfection, the cells were incubated with vehicle, 10 μM cyanidin, cyanidin plus 10 μM GW6471, or cyanidin plus 10 μM GW9662 for 6 h. The relative promoter activity was represented relative to the values in vehicle. n = 3–4. ** p
Figure Legend Snippet: Involvement of PPARα on the cyanidin-induced elevation of TRPM6 and CNNM4 promoter activities. TRPM6 promoter ( A ) or CNNM4 promoter ( B ) luciferase vectors were co-transfected with a pRL-TK vector into MCE301 cells. WT and mutant indicate vectors with wild-type and mutant PPRE sequences, respectively. At 24 h after transfection, the cells were incubated with vehicle, 10 μM cyanidin, cyanidin plus 10 μM GW6471, or cyanidin plus 10 μM GW9662 for 6 h. The relative promoter activity was represented relative to the values in vehicle. n = 3–4. ** p

Techniques Used: Luciferase, Transfection, Plasmid Preparation, Mutagenesis, Incubation, Activity Assay

2) Product Images from "Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities"

Article Title: Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki726

Cellular activity of (T,C)-containing TFO/LNAs: inhibition of transcription elongation. (A) Experimental design. Schematic representation of the (+)PPT/luc or (+)mutPPT/luc plasmids. These plasmids allow expression of the firefly luciferase reporter gene (luc) (Materials and Methods). The PPT (or the mutPPT 5′, AAAAGAAAA A GG A GGA-3′, two mutations in bold) sequence was inserted just upstream the luc gene. ( B ) The expression vector, (+)PPT/luc or (+)mutPPT/luc plasmid, was introduced with the pRL-TK plasmid, in the presence or absence of (T,C)-containing TFO/LNAs. Plasmids and TFOs were introduced independently using two transfection mixtures that were added to the cells at the same time. Cell extracts were analysed for firefly and Renilla luciferases activities, 24 h after transfection by a cationic dendrimer (Superfect). Normalized firefly/ Renilla luminescence intensity ratios are reported for different TFO/LNA concentrations. Data are derived from at least four experiments. Acr-16TC/po $ = Acr-16TC/po, 3′-modified by incorporation of a propylamine group. ( C ) Ratio of (Firefly/ Renilla ) RNA levels was deduced from competitive RT–PCR (Materials and Methods): decrease of RNA level was compared with that obtained for transfection of (+)PPT/luc and RL-TK plasmids in absence of TFO/LNAs.
Figure Legend Snippet: Cellular activity of (T,C)-containing TFO/LNAs: inhibition of transcription elongation. (A) Experimental design. Schematic representation of the (+)PPT/luc or (+)mutPPT/luc plasmids. These plasmids allow expression of the firefly luciferase reporter gene (luc) (Materials and Methods). The PPT (or the mutPPT 5′, AAAAGAAAA A GG A GGA-3′, two mutations in bold) sequence was inserted just upstream the luc gene. ( B ) The expression vector, (+)PPT/luc or (+)mutPPT/luc plasmid, was introduced with the pRL-TK plasmid, in the presence or absence of (T,C)-containing TFO/LNAs. Plasmids and TFOs were introduced independently using two transfection mixtures that were added to the cells at the same time. Cell extracts were analysed for firefly and Renilla luciferases activities, 24 h after transfection by a cationic dendrimer (Superfect). Normalized firefly/ Renilla luminescence intensity ratios are reported for different TFO/LNA concentrations. Data are derived from at least four experiments. Acr-16TC/po $ = Acr-16TC/po, 3′-modified by incorporation of a propylamine group. ( C ) Ratio of (Firefly/ Renilla ) RNA levels was deduced from competitive RT–PCR (Materials and Methods): decrease of RNA level was compared with that obtained for transfection of (+)PPT/luc and RL-TK plasmids in absence of TFO/LNAs.

Techniques Used: Activity Assay, Inhibition, Expressing, Luciferase, Sequencing, Plasmid Preparation, Transfection, Derivative Assay, Modification, Reverse Transcription Polymerase Chain Reaction

3) Product Images from "TNF-α treatment increases DKK1 protein levels in primary osteoblasts via upregulation of DKK1 mRNA levels and downregulation of miR-335-5p"

Article Title: TNF-α treatment increases DKK1 protein levels in primary osteoblasts via upregulation of DKK1 mRNA levels and downregulation of miR-335-5p

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2020.11152

TNF-α treatment exhibits no effects on the inhibitory action of miR-335-5p via targeting of DKK1 3′UTR. MC3T3-E1 cells were co-transfected with pMIR-REPORT-DKK1 UTR and pRL-TK Vector, and cells co-transfected with pMIR-REPORT and pRL-TK served as controls. (A) Transfected MC3T3-E1 cells were treated with 15 ng/ml TNF-α for 0, 12, 24 and 48 h, and the luciferase levels were determined. *P
Figure Legend Snippet: TNF-α treatment exhibits no effects on the inhibitory action of miR-335-5p via targeting of DKK1 3′UTR. MC3T3-E1 cells were co-transfected with pMIR-REPORT-DKK1 UTR and pRL-TK Vector, and cells co-transfected with pMIR-REPORT and pRL-TK served as controls. (A) Transfected MC3T3-E1 cells were treated with 15 ng/ml TNF-α for 0, 12, 24 and 48 h, and the luciferase levels were determined. *P

Techniques Used: Transfection, Plasmid Preparation, Luciferase

TNF-α treatment exhibits no effects on the inhibitory action of miR-335-5p via targeting of DKK1 3′UTR. MC3T3-E1 cells were co-transfected with pMIR-REPORT-DKK1 UTR and pRL-TK Vector, and cells co-transfected with pMIR-REPORT and pRL-TK served as controls. (A) Transfected MC3T3-E1 cells were treated with 15 ng/ml TNF-α for 0, 12, 24 and 48 h, and the luciferase levels were determined. *P
Figure Legend Snippet: TNF-α treatment exhibits no effects on the inhibitory action of miR-335-5p via targeting of DKK1 3′UTR. MC3T3-E1 cells were co-transfected with pMIR-REPORT-DKK1 UTR and pRL-TK Vector, and cells co-transfected with pMIR-REPORT and pRL-TK served as controls. (A) Transfected MC3T3-E1 cells were treated with 15 ng/ml TNF-α for 0, 12, 24 and 48 h, and the luciferase levels were determined. *P

Techniques Used: Transfection, Plasmid Preparation, Luciferase

4) Product Images from "Transcriptional activation of microRNA-34a by NF-kappa B in human esophageal cancer cells"

Article Title: Transcriptional activation of microRNA-34a by NF-kappa B in human esophageal cancer cells

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-13-4

P53 is necessary for NF-κB-induced miR-34a transcriptional activity but not for NF-κB binding . (a) KYSE450 cells were transfected with pCDNA3.1or p65 in combination with the wildtype miR-34a promoter (P1). pRL-TK was used as transfection control. Cells were lysised and luciferase assay were performed 48 h after transfection. Error bars represent the standard deviations for three independent experiments. (b) pCDNA3.1or p65 and siRNA control or sip53 along with the wildtype miR-34a promoter were cotransfected in EC109. pRL-TK vectors were used as transfection control. Luciferase assay were performed 48 h after transfection. Error bars represent the standard deviations for three independent experiments. (c) EMSA was performed with nuclear extracts from KYSE450 cells using probe corresponding to the third κB site (34a3ΚB) in miR-34a promoter region. Competition and supershift assays against anti-p53, p65 and p50 antibody were also shown. (d) Chromatin derived from KYSE450 cells transfected with p65 or control vectors were immunoprecipitated with anti-p65, p50, p53 and IgG antibodies. Relative enrichment of each transcription factor-bound DNA was detected by qPCR using 34aΚB3 primers. Shown were results normalizing to input DNA.
Figure Legend Snippet: P53 is necessary for NF-κB-induced miR-34a transcriptional activity but not for NF-κB binding . (a) KYSE450 cells were transfected with pCDNA3.1or p65 in combination with the wildtype miR-34a promoter (P1). pRL-TK was used as transfection control. Cells were lysised and luciferase assay were performed 48 h after transfection. Error bars represent the standard deviations for three independent experiments. (b) pCDNA3.1or p65 and siRNA control or sip53 along with the wildtype miR-34a promoter were cotransfected in EC109. pRL-TK vectors were used as transfection control. Luciferase assay were performed 48 h after transfection. Error bars represent the standard deviations for three independent experiments. (c) EMSA was performed with nuclear extracts from KYSE450 cells using probe corresponding to the third κB site (34a3ΚB) in miR-34a promoter region. Competition and supershift assays against anti-p53, p65 and p50 antibody were also shown. (d) Chromatin derived from KYSE450 cells transfected with p65 or control vectors were immunoprecipitated with anti-p65, p50, p53 and IgG antibodies. Relative enrichment of each transcription factor-bound DNA was detected by qPCR using 34aΚB3 primers. Shown were results normalizing to input DNA.

Techniques Used: Activity Assay, Binding Assay, Transfection, Luciferase, Derivative Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction

5) Product Images from "Selenium-binding protein 1 transcriptionally activates p21 expression via p53-independent mechanism and its frequent reduction associates with poor prognosis in bladder cancer"

Article Title: Selenium-binding protein 1 transcriptionally activates p21 expression via p53-independent mechanism and its frequent reduction associates with poor prognosis in bladder cancer

Journal: Journal of Translational Medicine

doi: 10.1186/s12967-020-02211-4

SELENBP1 transcriptionally stimulates p21 expression in a p53-independent manner. a Total RNA was isolated from indicated stable transfectants, and then subjected to RT-PCR assay for determining p21 mRNA levels. The mRNA levels of GAPDH were used as a loading control. b The potential binding sites of multiple transcription factors in the p21 promoter are shown in upper panel , and full-length (2.4 Kb), 1.8 Kb, 1.3 Kb and 200 bp of p21 promoter-driven luciferase reporters are schematically shown in lower panel . c Stable UMUC3 (Vector) and UMUC3 (HA-SELENBP1) cells were transiently co-transfected with pRL-TK and p21 promoter-driven luciferase reporters as described in “ b ”. Following 36 h of transient transfection, luciferase reporter activity was determined and normalized to that of corresponding UMUC3-Vector. The asterisk (*) indicates a significant difference in comparison to that of corresponding control transfectants ( p
Figure Legend Snippet: SELENBP1 transcriptionally stimulates p21 expression in a p53-independent manner. a Total RNA was isolated from indicated stable transfectants, and then subjected to RT-PCR assay for determining p21 mRNA levels. The mRNA levels of GAPDH were used as a loading control. b The potential binding sites of multiple transcription factors in the p21 promoter are shown in upper panel , and full-length (2.4 Kb), 1.8 Kb, 1.3 Kb and 200 bp of p21 promoter-driven luciferase reporters are schematically shown in lower panel . c Stable UMUC3 (Vector) and UMUC3 (HA-SELENBP1) cells were transiently co-transfected with pRL-TK and p21 promoter-driven luciferase reporters as described in “ b ”. Following 36 h of transient transfection, luciferase reporter activity was determined and normalized to that of corresponding UMUC3-Vector. The asterisk (*) indicates a significant difference in comparison to that of corresponding control transfectants ( p

Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay

The restoration of SELENBP1 leads to the suppression of c-Jun and STAT1 phosphorylation, both of which are required for SELENBP1-mediated transcriptional induction of p21 protein. a Cell lysates of stable UMUC3 (Vector) and UMUC3 (HA-SELENBP1) cells were subjected to immunoblotting assay with indicated antibodies that might be involved in transcriptional regulation of p21 expression. b UMUC3 cells stably transfected with dominant-negative mutant form of c-Jun (TAM67) or pcDNA3.1(Vector) were extracted and then subjected to immunoblotting analysis with indicated antibodies. Densitometric quantification of p21 (relative to GAPDH) is shown. c UMUC3 cells were stably transfected with dominant-negative mutant STAT1 (DN-STAT1) or pEGFP-C1 construct (Vector), and were then extracted for immunoblotting assay with indicated antibodies. Densitometric quantification of p21 and SOCS1 (relative to GAPDH) is shown under each blot. d TAM67 (1 μg) and pcDNA3.1 (1 μg) plasmids were transiently transfected into stable UMUC3 (DN-STAT1) and UMUC3 (pEGFP-C1), respectively. Following 48 h of transient transfection, cells were collected and then subjected to immunoblotting assay. Densitometric quantification of p21 (relative to GAPDH) is shown. e Indicated amounts of empty vector (pcDNA3.1) and TAM67 was transiently co-transfected into stable UMUC3 (DN-STAT1) or UMUC3 (Vector) cells, together with 1.3 Kb of p21 promoter-driven luciferase reporter and pRL-TK, as an internal control. Thirty-six hours post transfection, luciferase reporter activity was determined. Luciferase activities of UMUC3 (Vector) and UMUC3 (DN-STAT1) groups are normalized to those of corresponding control group that transfected with 0.6 μg of pcDNA3.1, respectively. The asterisk (*) indicates a significant difference as compared to UMUC3 (DN-STAT1) group transfected with 0.6 μg of pcDNA3.1 ( p
Figure Legend Snippet: The restoration of SELENBP1 leads to the suppression of c-Jun and STAT1 phosphorylation, both of which are required for SELENBP1-mediated transcriptional induction of p21 protein. a Cell lysates of stable UMUC3 (Vector) and UMUC3 (HA-SELENBP1) cells were subjected to immunoblotting assay with indicated antibodies that might be involved in transcriptional regulation of p21 expression. b UMUC3 cells stably transfected with dominant-negative mutant form of c-Jun (TAM67) or pcDNA3.1(Vector) were extracted and then subjected to immunoblotting analysis with indicated antibodies. Densitometric quantification of p21 (relative to GAPDH) is shown. c UMUC3 cells were stably transfected with dominant-negative mutant STAT1 (DN-STAT1) or pEGFP-C1 construct (Vector), and were then extracted for immunoblotting assay with indicated antibodies. Densitometric quantification of p21 and SOCS1 (relative to GAPDH) is shown under each blot. d TAM67 (1 μg) and pcDNA3.1 (1 μg) plasmids were transiently transfected into stable UMUC3 (DN-STAT1) and UMUC3 (pEGFP-C1), respectively. Following 48 h of transient transfection, cells were collected and then subjected to immunoblotting assay. Densitometric quantification of p21 (relative to GAPDH) is shown. e Indicated amounts of empty vector (pcDNA3.1) and TAM67 was transiently co-transfected into stable UMUC3 (DN-STAT1) or UMUC3 (Vector) cells, together with 1.3 Kb of p21 promoter-driven luciferase reporter and pRL-TK, as an internal control. Thirty-six hours post transfection, luciferase reporter activity was determined. Luciferase activities of UMUC3 (Vector) and UMUC3 (DN-STAT1) groups are normalized to those of corresponding control group that transfected with 0.6 μg of pcDNA3.1, respectively. The asterisk (*) indicates a significant difference as compared to UMUC3 (DN-STAT1) group transfected with 0.6 μg of pcDNA3.1 ( p

Techniques Used: Plasmid Preparation, Expressing, Stable Transfection, Transfection, Dominant Negative Mutation, Construct, Luciferase, Activity Assay

6) Product Images from "Autosomal Dominant Diabetes Arising From a Wolfram Syndrome 1 Mutation"

Article Title: Autosomal Dominant Diabetes Arising From a Wolfram Syndrome 1 Mutation

Journal: Diabetes

doi: 10.2337/db13-0571

Luciferase reporter assays in HEK293T cells transfected with the ERSE reporter together with control (pcDNA), wild-type WFS1 (WT), mutant c.937C > T WFS1 (p.His313Tyr), or mutant c.940T > C WFS1 (p.Trp314Arg) expression plasmid. Cells were untreated (UT) or treated with TG (10 nmol/L) for 6 h. Relative intensity of luciferase (Promega Dual-Luciferase Reporter Assay System) was then measured ( n = 3; values are mean ± SD). Transfections were normalized with the pRL-TK vector (Promega) as an internal control. Welch t test on log-transformed data was used for determining the significance between two treatments. *** P
Figure Legend Snippet: Luciferase reporter assays in HEK293T cells transfected with the ERSE reporter together with control (pcDNA), wild-type WFS1 (WT), mutant c.937C > T WFS1 (p.His313Tyr), or mutant c.940T > C WFS1 (p.Trp314Arg) expression plasmid. Cells were untreated (UT) or treated with TG (10 nmol/L) for 6 h. Relative intensity of luciferase (Promega Dual-Luciferase Reporter Assay System) was then measured ( n = 3; values are mean ± SD). Transfections were normalized with the pRL-TK vector (Promega) as an internal control. Welch t test on log-transformed data was used for determining the significance between two treatments. *** P

Techniques Used: Luciferase, Transfection, Mutagenesis, Expressing, Plasmid Preparation, Reporter Assay, Transformation Assay

7) Product Images from "MicroRNA-196 Represses Bach1 Protein and HCV Gene Expression in Human Hepatoma Cells Expressing Hepatitis C Viral Proteins"

Article Title: MicroRNA-196 Represses Bach1 Protein and HCV Gene Expression in Human Hepatoma Cells Expressing Hepatitis C Viral Proteins

Journal: Hepatology (Baltimore, Md.)

doi: 10.1002/hep.23401

Mutant miR-196 mimic does not alter the luciferase activity of Bach1 3′-UTR reporter but inhibits the luciferase activity of mutant Bach1 3′-UTR reporter (A) Four nucleotides mutation were introduced to the seed match sites of miR-196. (B) Effect of mutant miR-196 on Bach1 3′-UTR reporter activity. 9-13 cells were co-transfected with 0.4 μg/mL of pGL3-Bach1, 0.4 μg/mL of pRL-TK and with 0, 10, 20 or 50 nM mimic negative control, miR-196 mimic or mutant miR-196 for 48 h, firefly and renilla luciferase activities were measured using Dual Luciferase Assay System from Promega. Firefly luciferase activities were normalized to renilla luciferase activities and total protein, as indicated in Materials and Methods. Values for cells without microRNA transfection were set equal to 1. (C) Restoration of seed match between mutant miR-196 and mutant Bach1 3′-UTR are shown. (D) Inhibition of mutant Bach1 3′-UTR reporter activity by mutant miR-196 mimic. 9-13 cells were co-transfected with 0.4 μg/mL of mutant reporter (pGL3-Bach1-Mut), 0.4 μg/mL of pRL-TK, and with 0, 10, 20 or 50 nM mutant miR-196 or wild type miR-196 mimic for 48 h, firefly and renilla luciferase activities were measured. Firefly luciferase activities were normalized to renilla luciferase activities and total protein. Values for cells without microRNA transfection were set equal to 1. Data are presented as means ± SE, n=3. * differs from mimic negative control, P
Figure Legend Snippet: Mutant miR-196 mimic does not alter the luciferase activity of Bach1 3′-UTR reporter but inhibits the luciferase activity of mutant Bach1 3′-UTR reporter (A) Four nucleotides mutation were introduced to the seed match sites of miR-196. (B) Effect of mutant miR-196 on Bach1 3′-UTR reporter activity. 9-13 cells were co-transfected with 0.4 μg/mL of pGL3-Bach1, 0.4 μg/mL of pRL-TK and with 0, 10, 20 or 50 nM mimic negative control, miR-196 mimic or mutant miR-196 for 48 h, firefly and renilla luciferase activities were measured using Dual Luciferase Assay System from Promega. Firefly luciferase activities were normalized to renilla luciferase activities and total protein, as indicated in Materials and Methods. Values for cells without microRNA transfection were set equal to 1. (C) Restoration of seed match between mutant miR-196 and mutant Bach1 3′-UTR are shown. (D) Inhibition of mutant Bach1 3′-UTR reporter activity by mutant miR-196 mimic. 9-13 cells were co-transfected with 0.4 μg/mL of mutant reporter (pGL3-Bach1-Mut), 0.4 μg/mL of pRL-TK, and with 0, 10, 20 or 50 nM mutant miR-196 or wild type miR-196 mimic for 48 h, firefly and renilla luciferase activities were measured. Firefly luciferase activities were normalized to renilla luciferase activities and total protein. Values for cells without microRNA transfection were set equal to 1. Data are presented as means ± SE, n=3. * differs from mimic negative control, P

Techniques Used: Mutagenesis, Luciferase, Activity Assay, Transfection, Negative Control, Inhibition

miR-196 mimic represses the expression of a luciferase reporter containing Bach1 3′-UTR (A) The Bach1 3′-UTR contains two seed match sites (highlighted) for miR-196. (B) Schematic representation of pGL3-Bach1, the firefly luciferase ( f-luc ) reporter construct utilized. (C) Inhibition of the f-luc activities of pGL3-Bach1 3′-UTR reporter by miR-196 mimic. 9-13 cells were co-transfected with 0.4 μg/mL of pGL3-Bach1, 0.4 μg/mL of pRL-TK (renilla) and with 50 nM miR-196 mimic, miR-16 mimic or miRNA mimic negative control (MMNC) by Lipofectamine 2000 as indicated. 48 h after transfection, the luciferase reporter activities were measured using Dual Luciferase Assay System from Promega. Firefly luciferase activities were normalized to renilla luciferase activities and total protein, as indicated in Materials and Methods. Values for cells with a mock transfection were set equal to 1. Data are presented as means ± SE, n=3. * differs from negative control only, P
Figure Legend Snippet: miR-196 mimic represses the expression of a luciferase reporter containing Bach1 3′-UTR (A) The Bach1 3′-UTR contains two seed match sites (highlighted) for miR-196. (B) Schematic representation of pGL3-Bach1, the firefly luciferase ( f-luc ) reporter construct utilized. (C) Inhibition of the f-luc activities of pGL3-Bach1 3′-UTR reporter by miR-196 mimic. 9-13 cells were co-transfected with 0.4 μg/mL of pGL3-Bach1, 0.4 μg/mL of pRL-TK (renilla) and with 50 nM miR-196 mimic, miR-16 mimic or miRNA mimic negative control (MMNC) by Lipofectamine 2000 as indicated. 48 h after transfection, the luciferase reporter activities were measured using Dual Luciferase Assay System from Promega. Firefly luciferase activities were normalized to renilla luciferase activities and total protein, as indicated in Materials and Methods. Values for cells with a mock transfection were set equal to 1. Data are presented as means ± SE, n=3. * differs from negative control only, P

Techniques Used: Expressing, Luciferase, Construct, Inhibition, Transfection, Negative Control

miR-196 does not inhibit luciferase activity of reporter with mutant Bach1 3′-UTR (A) Four nucleotides in two seed match sites of Bach1 3′-UTR were replaced. (B) miR-196 mimic decreased the f-luc activity of pGL3-Bach1-WT but not pGL-Bach1-Mut reporter. 9-13 cells were co-transfected with 0.4 μg/mL of mutant pGL3-Bach1 or 0.4 μg/mL of pGL3-Bach1 and with 0.4 μg/mL of pRL-TK (renilla), and with miR-196 mimic (10–50 nM) as indicated in Materials and Methods, 48 h after transfection, the luciferase reporter activities were measured using Dual Luciferase Assay System from Promega, firefly luciferase activities were normalized to renilla luciferase activities and total protein. (C) miR-155 mimic decreased the f-luc activities of both pGL3-Bach1-WT and pGL-Bach1-Mut reporter. 9-13 cells were co-transfected with 0.4 μg/mL of mutant pGL3-Bach1 or 0.4 μg/mL of pGL3-Bach1 and with 0.4 μg/mL of pRL-TK (renilla), and with miR-155 mimic (10–50 nM). 48 h after transfection, the luciferase reporter activities were assayed as described in Materials and Methods. Values for cells with a mock transfection were set equal to 1. Data are presented as means ± SE, n=3. * differs from negative control only, P
Figure Legend Snippet: miR-196 does not inhibit luciferase activity of reporter with mutant Bach1 3′-UTR (A) Four nucleotides in two seed match sites of Bach1 3′-UTR were replaced. (B) miR-196 mimic decreased the f-luc activity of pGL3-Bach1-WT but not pGL-Bach1-Mut reporter. 9-13 cells were co-transfected with 0.4 μg/mL of mutant pGL3-Bach1 or 0.4 μg/mL of pGL3-Bach1 and with 0.4 μg/mL of pRL-TK (renilla), and with miR-196 mimic (10–50 nM) as indicated in Materials and Methods, 48 h after transfection, the luciferase reporter activities were measured using Dual Luciferase Assay System from Promega, firefly luciferase activities were normalized to renilla luciferase activities and total protein. (C) miR-155 mimic decreased the f-luc activities of both pGL3-Bach1-WT and pGL-Bach1-Mut reporter. 9-13 cells were co-transfected with 0.4 μg/mL of mutant pGL3-Bach1 or 0.4 μg/mL of pGL3-Bach1 and with 0.4 μg/mL of pRL-TK (renilla), and with miR-155 mimic (10–50 nM). 48 h after transfection, the luciferase reporter activities were assayed as described in Materials and Methods. Values for cells with a mock transfection were set equal to 1. Data are presented as means ± SE, n=3. * differs from negative control only, P

Techniques Used: Luciferase, Activity Assay, Mutagenesis, Transfection, Negative Control

8) Product Images from "Repression of Human T-lymphotropic virus type 1 Long Terminal Repeat sense transcription by Sp1 recruitment to novel Sp1 binding sites"

Article Title: Repression of Human T-lymphotropic virus type 1 Long Terminal Repeat sense transcription by Sp1 recruitment to novel Sp1 binding sites

Journal: Scientific Reports

doi: 10.1038/srep43221

Comparison of the Tax inductibility of the HTLV-1 promoters in non episomal and episomal reporter vectors. Jurkat T cells were transiently cotransfected with 600 ng of either pREP-LTRwt_sense-luc (( a ) lanes 1 to 6), pREP∆-LTRwt_sense-luc (( a ) lanes 7 to 12), pREP-LTRwt_antisense-luc (( b ) lanes 1 to 6) or pREP∆-LTRwt_antisense-luc (( b ) lanes 7 to 12) and increasing amounts of a HTLV-1 Tax expression vector pRSV-Tax (0, 25, 50, 100, 200 and 400 ng of plasmid DNA). To maintain the same amount of transfected DNA and to avoid squelching artifacts, the different amounts of pRSV-Tax cotransfected were complemented to 400 ng of DNA by using the corresponding empty plasmid. Each transfection included 10 ng of the internal control Renilla luciferase plasmid, pRL-TK. Luciferase activities (Firefly and Renilla ) and protein concentrations were measured in cell lysates 48 h after transfection. The results are expressed as luciferase firefly /luciferase Renilla /[protein] and presented as histograms indicating the induction by Tax (in fold) with respect to the activity of each LTR reporter construct in the absence of Tax, which was arbitrarily assigned a value of 1. Means and standard errors of the means from triplicate samples are presented. An experiment representative of three independent experiments is shown. ns corresponds to a p value > 0.05, ***Corresponds to a p value
Figure Legend Snippet: Comparison of the Tax inductibility of the HTLV-1 promoters in non episomal and episomal reporter vectors. Jurkat T cells were transiently cotransfected with 600 ng of either pREP-LTRwt_sense-luc (( a ) lanes 1 to 6), pREP∆-LTRwt_sense-luc (( a ) lanes 7 to 12), pREP-LTRwt_antisense-luc (( b ) lanes 1 to 6) or pREP∆-LTRwt_antisense-luc (( b ) lanes 7 to 12) and increasing amounts of a HTLV-1 Tax expression vector pRSV-Tax (0, 25, 50, 100, 200 and 400 ng of plasmid DNA). To maintain the same amount of transfected DNA and to avoid squelching artifacts, the different amounts of pRSV-Tax cotransfected were complemented to 400 ng of DNA by using the corresponding empty plasmid. Each transfection included 10 ng of the internal control Renilla luciferase plasmid, pRL-TK. Luciferase activities (Firefly and Renilla ) and protein concentrations were measured in cell lysates 48 h after transfection. The results are expressed as luciferase firefly /luciferase Renilla /[protein] and presented as histograms indicating the induction by Tax (in fold) with respect to the activity of each LTR reporter construct in the absence of Tax, which was arbitrarily assigned a value of 1. Means and standard errors of the means from triplicate samples are presented. An experiment representative of three independent experiments is shown. ns corresponds to a p value > 0.05, ***Corresponds to a p value

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

Ability of multimerized HTLV-1 Sp1#3 and Sp1#4 putative binding sites to confer Sp1 stimulation to a TK minimal promoter in Drosophila SL-2 cell line. SL-2 cells were cotransfected with 100 ng of a TK luciferase reporter construct devoid (lanes 1 to 4) or not of upstream wild-type or mutated Sp1#3 and Sp1#4 motifs (as indicated, lanes 5 to 20) and increasing amounts (0, 25, 50 and 100 ng) of an Sp1 expression vector (pPacSp1). To maintain the same amount of transfected DNA and to avoid squelching artifacts, the different amounts of pPacSp1 cotransfected were complemented to 100 ng of DNA by using the corresponding empty plasmid. Each transfection included 5 ng of the internal control plasmid, pRL-TK, in which the herpes simplex virus thymidine kinase promoter drives Renilla luciferase gene expression. Luciferase activities (Firefly and Renilla ) were measured in cell lysates 24 h after transfection. The results are expressed as luciferase firefly /luciferase Renilla and presented as histograms indicating the induction by Sp1 (in fold) with respect to the activity of each TK reporter construct in the absence of Sp1, which was assigned a value of 1. Means and standard errors of the means from triplicate samples are represented. An experiment representative of three independent experiments is shown. ns corresponds to a p value > 0.05, ***Corresponds to a p value
Figure Legend Snippet: Ability of multimerized HTLV-1 Sp1#3 and Sp1#4 putative binding sites to confer Sp1 stimulation to a TK minimal promoter in Drosophila SL-2 cell line. SL-2 cells were cotransfected with 100 ng of a TK luciferase reporter construct devoid (lanes 1 to 4) or not of upstream wild-type or mutated Sp1#3 and Sp1#4 motifs (as indicated, lanes 5 to 20) and increasing amounts (0, 25, 50 and 100 ng) of an Sp1 expression vector (pPacSp1). To maintain the same amount of transfected DNA and to avoid squelching artifacts, the different amounts of pPacSp1 cotransfected were complemented to 100 ng of DNA by using the corresponding empty plasmid. Each transfection included 5 ng of the internal control plasmid, pRL-TK, in which the herpes simplex virus thymidine kinase promoter drives Renilla luciferase gene expression. Luciferase activities (Firefly and Renilla ) were measured in cell lysates 24 h after transfection. The results are expressed as luciferase firefly /luciferase Renilla and presented as histograms indicating the induction by Sp1 (in fold) with respect to the activity of each TK reporter construct in the absence of Sp1, which was assigned a value of 1. Means and standard errors of the means from triplicate samples are represented. An experiment representative of three independent experiments is shown. ns corresponds to a p value > 0.05, ***Corresponds to a p value

Techniques Used: Binding Assay, Luciferase, Construct, Expressing, Plasmid Preparation, Transfection, Activity Assay

Effects of HTLV-1 Sp1-binding sites mutations on basal and Tax-transactivated 5′-LTR promoter activity. Using site-directed mutagenesis, we generated HTLV-1 sense LTR promoter luciferase episomal constructs containing 2-bp mutations abolishing Sp1 binding to the LTR Sp1 binding sites, alone or in combination (see Supplementary Table S2 for constructs description). These generated plasmids (600 ng of DNA) were cotransfected, with 400 ng of the pRSV empty vector (+pRSV) or with 400 ng of the Tax expression vector (+pRSV-Tax) into Jurkat T cells. Each transfection included 10 ng of the internal control Renilla luciferase plasmid, pRL-TK. Luciferase activities (Firefly and Renilla ) and protein concentrations were measured in cell lysates 48 h after transfection. The results are expressed as luciferase firefly /luciferase Renilla /[protein] and presented as histograms indicating luciferase activity (arbitrary unit presented in ( a )) of each reporter construct and relative to that measured with the pREP-LTRwt_sense-luc (LTRwt_s), which was arbitrarily assigned a value of 1, in the absence of the Tax transactivator (presented in ( b )). Induction by Tax (in fold) with respect to the activity of the same reporter construct in the absence of Tax, which was arbitrarily assigned a value of 1 are presented in ( c ). Means and standard errors of the means from triplicate samples are represented. An experiment representative of three independent experiments is shown. ns corresponds to a p value > 0.05, *Corresponds to a p value
Figure Legend Snippet: Effects of HTLV-1 Sp1-binding sites mutations on basal and Tax-transactivated 5′-LTR promoter activity. Using site-directed mutagenesis, we generated HTLV-1 sense LTR promoter luciferase episomal constructs containing 2-bp mutations abolishing Sp1 binding to the LTR Sp1 binding sites, alone or in combination (see Supplementary Table S2 for constructs description). These generated plasmids (600 ng of DNA) were cotransfected, with 400 ng of the pRSV empty vector (+pRSV) or with 400 ng of the Tax expression vector (+pRSV-Tax) into Jurkat T cells. Each transfection included 10 ng of the internal control Renilla luciferase plasmid, pRL-TK. Luciferase activities (Firefly and Renilla ) and protein concentrations were measured in cell lysates 48 h after transfection. The results are expressed as luciferase firefly /luciferase Renilla /[protein] and presented as histograms indicating luciferase activity (arbitrary unit presented in ( a )) of each reporter construct and relative to that measured with the pREP-LTRwt_sense-luc (LTRwt_s), which was arbitrarily assigned a value of 1, in the absence of the Tax transactivator (presented in ( b )). Induction by Tax (in fold) with respect to the activity of the same reporter construct in the absence of Tax, which was arbitrarily assigned a value of 1 are presented in ( c ). Means and standard errors of the means from triplicate samples are represented. An experiment representative of three independent experiments is shown. ns corresponds to a p value > 0.05, *Corresponds to a p value

Techniques Used: Binding Assay, Activity Assay, Mutagenesis, Generated, Luciferase, Construct, Plasmid Preparation, Expressing, Transfection

Effects of HTLV-1 Sp1-binding sites mutations on basal and Tax-transactivated 3′-LTR promoter activity. Using site-directed mutagenesis, we generated HTLV-1 antisense LTR promoter luciferase episomal constructs containing 2-bp mutations abolishing Sp1 binding to the LTR Sp1 binding sites, alone or in combination (see Supplementary Table S2 for constructs description). These generated plamsids (600 ng of DNA) were cotransfected, with 400 ng of the pRSV empty vector (+pRSV) or with 400 ng of the Tax expression vector (+pRSV-Tax) into Jurkat T cells. Each transfection included 10 ng of the internal control Renilla luciferase plasmid, pRL-TK. Luciferase activities (Firefly and Renilla ) and protein concentrations were measured in cell lysates 48 h after transfection. The results are expressed as luciferase firefly /luciferase Renilla /[protein] and presented as histograms indicating luciferase activity (arbitrary unit presented in ( a )). of each reporter construct and relative to that measured with the pREP-LTRwt_antisense-luc (LTRwt_as), which was arbitrarily assigned a value of 1, in the absence of the Tax transactivator (presented in ( b )). Induction by Tax (in fold) with respect to the activity of the same reporter construct in the absence of Tax, which was arbitrarily assigned a value of 1 are presented in c . Means and standard errors of the means from triplicate samples are represented. An experiment representative of three independent experiments is shown. ns corresponds to a p value > 0.05, *Corresponds to a p value
Figure Legend Snippet: Effects of HTLV-1 Sp1-binding sites mutations on basal and Tax-transactivated 3′-LTR promoter activity. Using site-directed mutagenesis, we generated HTLV-1 antisense LTR promoter luciferase episomal constructs containing 2-bp mutations abolishing Sp1 binding to the LTR Sp1 binding sites, alone or in combination (see Supplementary Table S2 for constructs description). These generated plamsids (600 ng of DNA) were cotransfected, with 400 ng of the pRSV empty vector (+pRSV) or with 400 ng of the Tax expression vector (+pRSV-Tax) into Jurkat T cells. Each transfection included 10 ng of the internal control Renilla luciferase plasmid, pRL-TK. Luciferase activities (Firefly and Renilla ) and protein concentrations were measured in cell lysates 48 h after transfection. The results are expressed as luciferase firefly /luciferase Renilla /[protein] and presented as histograms indicating luciferase activity (arbitrary unit presented in ( a )). of each reporter construct and relative to that measured with the pREP-LTRwt_antisense-luc (LTRwt_as), which was arbitrarily assigned a value of 1, in the absence of the Tax transactivator (presented in ( b )). Induction by Tax (in fold) with respect to the activity of the same reporter construct in the absence of Tax, which was arbitrarily assigned a value of 1 are presented in c . Means and standard errors of the means from triplicate samples are represented. An experiment representative of three independent experiments is shown. ns corresponds to a p value > 0.05, *Corresponds to a p value

Techniques Used: Binding Assay, Activity Assay, Mutagenesis, Generated, Luciferase, Construct, Plasmid Preparation, Expressing, Transfection

9) Product Images from "Influenza A virus-induced downregulation of miR-26a contributes to reduced IFNα/β production"

Article Title: Influenza A virus-induced downregulation of miR-26a contributes to reduced IFNα/β production

Journal: Virologica Sinica

doi: 10.1007/s12250-017-4004-9

miR-26a targets USP3. (A) 293T cells were co-transfected with the plasmids encoding RIG-I, VISA, TBK1 and IRF3 (200 ng), reporter vector pIFN-β-Luc (200 ng), inter control vector pRL-TK (20 ng) with or without miR-26a mimic (100 nmol/L). At 30 h post-transfection, luciferase activity was measured. (B) Sequence complementarity between miR-26a and the 3κUTR of USP3 gene. (C) 293T cells were transfected with wild type or mutant USP3 3κUTR-luc reporter vector (400 ng), pGL3-control vector (20 ng) along with miR-26a or miR negative control for 36 h. The cells were harvested, and a luciferase assay was performed. (D) USP3 protein levels in 293T cells were analyzed by western blotting after transfection with miR-26a (100 nmol/L) or miR-26a inhibitor (100 nmol/L) for 48 h followed by infection with A/WSN/33 virus (MOI = 1) for another 4 h. (E) 293T cells were transfected with the plasmid encoding Myc-USP3 (500 ng) for 24 h followed by infection with A/WSN/33 virus for another 16 h. The supernatants of the cell cultures were harvested and measured by plaque assays as the viral titers and the cell lysates were analyzed by western blotting to confirm the expression of Myc-USP3. (F) 293T cells were infected with A/WSN/33 virus (MOI = 1) followed by treatment with Bay11 (10 µmol/L) for 4 h or left untreated. miR-26a expression was measured by real-time qRT-PCR and USP3 protein levels were analyzed by western blotting. Data represent mean ± S.D. of three independent experiments. ns, no significant difference.
Figure Legend Snippet: miR-26a targets USP3. (A) 293T cells were co-transfected with the plasmids encoding RIG-I, VISA, TBK1 and IRF3 (200 ng), reporter vector pIFN-β-Luc (200 ng), inter control vector pRL-TK (20 ng) with or without miR-26a mimic (100 nmol/L). At 30 h post-transfection, luciferase activity was measured. (B) Sequence complementarity between miR-26a and the 3κUTR of USP3 gene. (C) 293T cells were transfected with wild type or mutant USP3 3κUTR-luc reporter vector (400 ng), pGL3-control vector (20 ng) along with miR-26a or miR negative control for 36 h. The cells were harvested, and a luciferase assay was performed. (D) USP3 protein levels in 293T cells were analyzed by western blotting after transfection with miR-26a (100 nmol/L) or miR-26a inhibitor (100 nmol/L) for 48 h followed by infection with A/WSN/33 virus (MOI = 1) for another 4 h. (E) 293T cells were transfected with the plasmid encoding Myc-USP3 (500 ng) for 24 h followed by infection with A/WSN/33 virus for another 16 h. The supernatants of the cell cultures were harvested and measured by plaque assays as the viral titers and the cell lysates were analyzed by western blotting to confirm the expression of Myc-USP3. (F) 293T cells were infected with A/WSN/33 virus (MOI = 1) followed by treatment with Bay11 (10 µmol/L) for 4 h or left untreated. miR-26a expression was measured by real-time qRT-PCR and USP3 protein levels were analyzed by western blotting. Data represent mean ± S.D. of three independent experiments. ns, no significant difference.

Techniques Used: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Sequencing, Mutagenesis, Negative Control, Western Blot, Infection, Expressing, Quantitative RT-PCR

miR-26a does not directly target IAV genome but instead activates IFN signal pathway. (A) Fragments of IAV genome were inserted into the 3′UTR of the luciferase gene in pRL-TK vector as indicated. (B) miR-26a mimic (100 nmol/L) and the constructs (400 ng) were co-transfected into 293T cells. The luciferase activity assays were performed at 36 h post transfection. (C) 293T cells were co-transfected with miR-26a mimic (100 nmol/L), reporter vector pNF-κB-Luc (400 ng), pIFN-β-Luc (400 ng), or pISRE-Luc (400 ng), with pRL-TK (20 ng) as an internal control. At 24 h post-transfection, the cells were infected with A/WSN/33 virus (MOI = 1) or left uninfected. Then the luciferase assays were performed at 12 hpi. (D) 293T cells were transfected with miR-26a mimic (100 nmol/L) or inhibitor (100 nmol/L) followed by infection with A/WSN/33 virus. IFN-α, IFN-β, ISG15 and IL8 mRNA expression were measured by qRT-PCR analysis at 8 hpi. IFN-β levels in the supernatants were measured by ELISA at 16 hpi (E). Data represent mean ± S.D. of three independent experiments.* P
Figure Legend Snippet: miR-26a does not directly target IAV genome but instead activates IFN signal pathway. (A) Fragments of IAV genome were inserted into the 3′UTR of the luciferase gene in pRL-TK vector as indicated. (B) miR-26a mimic (100 nmol/L) and the constructs (400 ng) were co-transfected into 293T cells. The luciferase activity assays were performed at 36 h post transfection. (C) 293T cells were co-transfected with miR-26a mimic (100 nmol/L), reporter vector pNF-κB-Luc (400 ng), pIFN-β-Luc (400 ng), or pISRE-Luc (400 ng), with pRL-TK (20 ng) as an internal control. At 24 h post-transfection, the cells were infected with A/WSN/33 virus (MOI = 1) or left uninfected. Then the luciferase assays were performed at 12 hpi. (D) 293T cells were transfected with miR-26a mimic (100 nmol/L) or inhibitor (100 nmol/L) followed by infection with A/WSN/33 virus. IFN-α, IFN-β, ISG15 and IL8 mRNA expression were measured by qRT-PCR analysis at 8 hpi. IFN-β levels in the supernatants were measured by ELISA at 16 hpi (E). Data represent mean ± S.D. of three independent experiments.* P

Techniques Used: Luciferase, Plasmid Preparation, Construct, Transfection, Activity Assay, Infection, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

IAV infection induces down-regulation of miR-26a in cells via NF-κB pathway. (A) 293T cells were infected with A/WSN/33 virus at an MOI of 1 and A549 cells were infected at an MOI of 2 for the indicated time. Expression of miR-26a was evaluated by qRT-PCR with U6 as an internal control, NP expression was measured by Western blot to confirm the successful infection and replication of A/WSN/33 virus at 293T and A549 cells. (B) 293T and A549 cells were infected with A/WSN/33 virus at the indicated MOI for 8 h. Expression of miR-26a was evaluated as described in (A), (C) IAV infection down-regulates miR-26a expression mainly through NF-κB pathway. (C-a) 293T cells were trans-fected with pNF-κB-Luc (400 ng) and pRL-TK (20 ng), which used as an internal control for 24 h followed by infection with A/WSN/33 virus (MOI=1) and incubation with or without NF-κB inhibitor Bay11 for 4 h. At 16 h post-infection, cells were lysed for luciferase activity assay. (C-b) 293T cells were infected with A/WSN/33 virus (MOI=1) followed by treatment with Bay11 (10 µmol/L) for 4 h or left untreated. At 8 h post-infection, miR-26a expression was measured as in (A). (C-c) 293T cells were transfected with the plasmid encoding p65 (500 ng) for 24 h and miR-26a expression was measured. Data represent mean ± S.D. of three independent experiments. * P
Figure Legend Snippet: IAV infection induces down-regulation of miR-26a in cells via NF-κB pathway. (A) 293T cells were infected with A/WSN/33 virus at an MOI of 1 and A549 cells were infected at an MOI of 2 for the indicated time. Expression of miR-26a was evaluated by qRT-PCR with U6 as an internal control, NP expression was measured by Western blot to confirm the successful infection and replication of A/WSN/33 virus at 293T and A549 cells. (B) 293T and A549 cells were infected with A/WSN/33 virus at the indicated MOI for 8 h. Expression of miR-26a was evaluated as described in (A), (C) IAV infection down-regulates miR-26a expression mainly through NF-κB pathway. (C-a) 293T cells were trans-fected with pNF-κB-Luc (400 ng) and pRL-TK (20 ng), which used as an internal control for 24 h followed by infection with A/WSN/33 virus (MOI=1) and incubation with or without NF-κB inhibitor Bay11 for 4 h. At 16 h post-infection, cells were lysed for luciferase activity assay. (C-b) 293T cells were infected with A/WSN/33 virus (MOI=1) followed by treatment with Bay11 (10 µmol/L) for 4 h or left untreated. At 8 h post-infection, miR-26a expression was measured as in (A). (C-c) 293T cells were transfected with the plasmid encoding p65 (500 ng) for 24 h and miR-26a expression was measured. Data represent mean ± S.D. of three independent experiments. * P

Techniques Used: Infection, Expressing, Quantitative RT-PCR, Western Blot, Incubation, Luciferase, Activity Assay, Transfection, Plasmid Preparation

10) Product Images from "Up-regulation of claudin-2 expression by aldosterone in colonic epithelial cells of mice fed with NaCl-depleted diets"

Article Title: Up-regulation of claudin-2 expression by aldosterone in colonic epithelial cells of mice fed with NaCl-depleted diets

Journal: Scientific Reports

doi: 10.1038/s41598-017-12494-1

Binding of MR to claudin-2 promoter by ALD. ( A ) Promoter luciferase constructs of claudin-2 including wild type (WT), mutant of SRE-1 (M1), and mutant of SRE-2 (M2) were co-transfected with pRL-TK vector into the cells. After 40 h of transfection, the cells were incubated in the presence and absence of 50 nM ALD for additional 8 h. The relative promoter activity was represented as the percent of WT in the absence of ALD. ( B ) Nuclear proteins were prepared from the cells treated with 50 nM ALD for 1 h in the presence and absence of 10 μM spironolactone (Spi) or 10 μM RU-486 (RU). Control cells (Cont) were not treated with these drugs. Vehicle (Veh) was treated with dimethylsulfoxide. After immunoprecipitation of genomic DNA by anti-MR antibody or rabbit IgG, semi-quantitative PCR was performed using the primers amplifying the MR binding site of claudin-2 promoter. Input chromatin was used for loading control. Statistical comparison was made by t test. ** P
Figure Legend Snippet: Binding of MR to claudin-2 promoter by ALD. ( A ) Promoter luciferase constructs of claudin-2 including wild type (WT), mutant of SRE-1 (M1), and mutant of SRE-2 (M2) were co-transfected with pRL-TK vector into the cells. After 40 h of transfection, the cells were incubated in the presence and absence of 50 nM ALD for additional 8 h. The relative promoter activity was represented as the percent of WT in the absence of ALD. ( B ) Nuclear proteins were prepared from the cells treated with 50 nM ALD for 1 h in the presence and absence of 10 μM spironolactone (Spi) or 10 μM RU-486 (RU). Control cells (Cont) were not treated with these drugs. Vehicle (Veh) was treated with dimethylsulfoxide. After immunoprecipitation of genomic DNA by anti-MR antibody or rabbit IgG, semi-quantitative PCR was performed using the primers amplifying the MR binding site of claudin-2 promoter. Input chromatin was used for loading control. Statistical comparison was made by t test. ** P

Techniques Used: Binding Assay, Luciferase, Construct, Mutagenesis, Transfection, Plasmid Preparation, Incubation, Activity Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction

11) Product Images from "2′-O-Galloylhyperin Isolated From Pyrola incarnata Fisch. Attenuates LPS-Induced Inflammatory Response by Activation of SIRT1/Nrf2 and Inhibition of the NF-κB Pathways in Vitro and Vivo"

Article Title: 2′-O-Galloylhyperin Isolated From Pyrola incarnata Fisch. Attenuates LPS-Induced Inflammatory Response by Activation of SIRT1/Nrf2 and Inhibition of the NF-κB Pathways in Vitro and Vivo

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00679

Effects of 2′- O -GH on LPS-induced activation of NF-κB and MAPKs pathway in RAW 264.7 macrophages cells. RAW264.7 cells were pretreated with 2′- O -GH for 2 h or left untreated and then with/without LPS (100 ng/mL) for 15 min (A) , 1 h (B–D) or 30 min (E) . (A) Lysates were analyzed by Western blot using antibodies against p-IκB-α, IκB-α, and p-IKKα/β. (B) Nuclear and cytosol extracts were subjected to Western blot analysis to determine the level of p65. Histone H3 was used as nuclear marker and β-actin as cytosol protein marker for standardization. (C) The translocation of p65 to the nucleus was analyzed by confocal microscopy. Cells were immunostained using FITC for p65 and DAPI for nucleus. (D) Cells were transfected with a pNF-κB-luc reporter vector and the pRL-TK vector as internal control. After 24 h of transfection, cells were treated with 2′- O -GH for 2 h and then stimulated with LPS (100 ng/mL) for 12 h. Cells were then harvested, and luciferase activity levels were determined as described in the Experimental Section. (E) Whole cell lysates were analyzed by Western blot using antibodies against activated MAPKs. Quantitative data were presented as mean ± SEM ( n = 3). ∗∗ p
Figure Legend Snippet: Effects of 2′- O -GH on LPS-induced activation of NF-κB and MAPKs pathway in RAW 264.7 macrophages cells. RAW264.7 cells were pretreated with 2′- O -GH for 2 h or left untreated and then with/without LPS (100 ng/mL) for 15 min (A) , 1 h (B–D) or 30 min (E) . (A) Lysates were analyzed by Western blot using antibodies against p-IκB-α, IκB-α, and p-IKKα/β. (B) Nuclear and cytosol extracts were subjected to Western blot analysis to determine the level of p65. Histone H3 was used as nuclear marker and β-actin as cytosol protein marker for standardization. (C) The translocation of p65 to the nucleus was analyzed by confocal microscopy. Cells were immunostained using FITC for p65 and DAPI for nucleus. (D) Cells were transfected with a pNF-κB-luc reporter vector and the pRL-TK vector as internal control. After 24 h of transfection, cells were treated with 2′- O -GH for 2 h and then stimulated with LPS (100 ng/mL) for 12 h. Cells were then harvested, and luciferase activity levels were determined as described in the Experimental Section. (E) Whole cell lysates were analyzed by Western blot using antibodies against activated MAPKs. Quantitative data were presented as mean ± SEM ( n = 3). ∗∗ p

Techniques Used: Activation Assay, Western Blot, Marker, Translocation Assay, Confocal Microscopy, Transfection, Plasmid Preparation, Luciferase, Activity Assay

12) Product Images from "2′‐Hydroxycinnamaldehyde inhibits proliferation and induces apoptosis via signal transducer and activator of transcription 3 inactivation and reactive oxygen species generation, et al. 2′‐Hydroxycinnamaldehyde inhibits proliferation and induces apoptosis via signal transducer and activator of transcription 3 inactivation and reactive oxygen species generation"

Article Title: 2′‐Hydroxycinnamaldehyde inhibits proliferation and induces apoptosis via signal transducer and activator of transcription 3 inactivation and reactive oxygen species generation, et al. 2′‐Hydroxycinnamaldehyde inhibits proliferation and induces apoptosis via signal transducer and activator of transcription 3 inactivation and reactive oxygen species generation

Journal: Cancer Science

doi: 10.1111/cas.13852

2′‐Hydroxycinnamaldehyde (HCA) suppresses cell proliferation via regulation of signal transducer and activator of transcription 3 (STAT3) activity in DU145 cells. A, The chemical structure of HCA. B, Luciferase activities were determined in DU145 cells that were co‐transfected with the 21pSTAT3‐TA‐Luc reporter vector and pRL‐TK vector for 6 h and subsequently treated with HCA for 24 h (n = 3). C, DU145 cells were treated with HCA for 24 or 48 h. Cell proliferation was measured by WST‐1 (n = 3). D, DU145 cells were treated with indicated concentrations of HCA for 24 h, and the expression level of p‐STAT (Y705) and STAT3 was analyzed by western blotting (n = 2). E, DU145 cells were treated with HCA (20 μmol/L) for the indicated times, and the expression level of p‐STAT (Y705) and STAT3 was analyzed by western blotting (n = 2). The band intensity of each protein was quantified using the MultiGauge program. * P
Figure Legend Snippet: 2′‐Hydroxycinnamaldehyde (HCA) suppresses cell proliferation via regulation of signal transducer and activator of transcription 3 (STAT3) activity in DU145 cells. A, The chemical structure of HCA. B, Luciferase activities were determined in DU145 cells that were co‐transfected with the 21pSTAT3‐TA‐Luc reporter vector and pRL‐TK vector for 6 h and subsequently treated with HCA for 24 h (n = 3). C, DU145 cells were treated with HCA for 24 or 48 h. Cell proliferation was measured by WST‐1 (n = 3). D, DU145 cells were treated with indicated concentrations of HCA for 24 h, and the expression level of p‐STAT (Y705) and STAT3 was analyzed by western blotting (n = 2). E, DU145 cells were treated with HCA (20 μmol/L) for the indicated times, and the expression level of p‐STAT (Y705) and STAT3 was analyzed by western blotting (n = 2). The band intensity of each protein was quantified using the MultiGauge program. * P

Techniques Used: High Content Screening, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Expressing, Western Blot

13) Product Images from "lncRNA SNHG1 Promotes Basal Bladder Cancer Invasion via Interaction with PP2A Catalytic Subunit and Induction of Autophagy"

Article Title: lncRNA SNHG1 Promotes Basal Bladder Cancer Invasion via Interaction with PP2A Catalytic Subunit and Induction of Autophagy

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2020.06.010

c-Jun Activation Mediated SNHG1-Promoted MMP2 Transcription in Human Basal MIBC Cells (A and B) MMP2 mRNA levels were evaluated in indicated transfectants using real-time PCR; GAPDH was used as an internal control. ∗p = 0.0085 in (A), and ∗p = 0.0073 in (B) (significant difference). (C and D) Indicated cells were transfected with MMP2 promoter-driven luciferase reporter together with pRL-TK. Transfectants were seeded into 96-well plates and then subjected to determine MMP2 promoter activity by measuring luciferase activity. pRL-TK was used as an internal control to normalize transfection efficiency. ∗p = 0.0088 in (C), and ∗p = 0.0081 in (D) (significant difference). (E) Indicated cells were seeded into 6-well plates. After synchronization, cells were treated with Act D for indicated times. Total RNA was then isolated and subjected to real-time PCR analysis of MMP2 mRNA levels; GAPDH was internal control. ∗p = 0.0095 (significant difference). (F) Indicated cell extracts underwent western blotting for determination of SP1, P50, p-P65, P65, p-c-Jun, and c-Jun expression. (G) TAM67 was stably transfected into U5637(SNHG1) cells, and the stable transfectants were then identified for c-Jun(D) (TAM67) expression and determination of MMP2; GAPDH was used as an internal control. (H) Indicated cells were extracted for total RNA with TRIzol. Real-time PCR was used to determine MMP2 mRNA expression; GAPDH was used as an internal control. Results shown are means ± SD from at least triplicate experiments. ∗p = 0.0005 (significant difference).
Figure Legend Snippet: c-Jun Activation Mediated SNHG1-Promoted MMP2 Transcription in Human Basal MIBC Cells (A and B) MMP2 mRNA levels were evaluated in indicated transfectants using real-time PCR; GAPDH was used as an internal control. ∗p = 0.0085 in (A), and ∗p = 0.0073 in (B) (significant difference). (C and D) Indicated cells were transfected with MMP2 promoter-driven luciferase reporter together with pRL-TK. Transfectants were seeded into 96-well plates and then subjected to determine MMP2 promoter activity by measuring luciferase activity. pRL-TK was used as an internal control to normalize transfection efficiency. ∗p = 0.0088 in (C), and ∗p = 0.0081 in (D) (significant difference). (E) Indicated cells were seeded into 6-well plates. After synchronization, cells were treated with Act D for indicated times. Total RNA was then isolated and subjected to real-time PCR analysis of MMP2 mRNA levels; GAPDH was internal control. ∗p = 0.0095 (significant difference). (F) Indicated cell extracts underwent western blotting for determination of SP1, P50, p-P65, P65, p-c-Jun, and c-Jun expression. (G) TAM67 was stably transfected into U5637(SNHG1) cells, and the stable transfectants were then identified for c-Jun(D) (TAM67) expression and determination of MMP2; GAPDH was used as an internal control. (H) Indicated cells were extracted for total RNA with TRIzol. Real-time PCR was used to determine MMP2 mRNA expression; GAPDH was used as an internal control. Results shown are means ± SD from at least triplicate experiments. ∗p = 0.0005 (significant difference).

Techniques Used: Activation Assay, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Activity Assay, Isolation, Western Blot, Expressing, Stable Transfection

miR-34a Downregulation Was Crucial for SNHG1-Mediated MMP2 mRNA Stabilization and Invasion in Basal MIBC Cells (A) An MMP2 mRNA 3′ UTR luciferase reporter was transiently transfected into the indicated cells, and the luciferase activity of each transfectant was evaluated. Luciferase activity is presented as relative to vector transfectant with normalization to internal transfection control pRL-TK. ∗p = 0.0083 (significant difference). (B) Quantitative real-time PCR was used to determine expression of miRNA in indicated cells. ∗p = 0.0091 (significant difference). (C) Schematic of construction of the MMP2 mRNA 3′ UTR luciferase reporter and its mutants were aligned with miR-34a. (D) WT and mutant MMP2 mRNA 3′ UTR luciferase reporters were transiently co-transfected with pRL-TK into indicated cells. Luciferase activity of each transfectant was evaluated; results are presented as relative MMP2 mRNA 3′ UTR activity. ∗p = 0.0063 (significant difference). (E) miR-34a constitutively expressed plasmids were stably transfected into U5637(SNHG1) cells. Stable transfectants were identified by real-time PCR. ∗p = 0.0065 (significant difference). (F) U5637(SNHG1/miR-34a) cells and their scramble vector transfectant were seeded into 6-well plates. After synchronization, cells were treated with Act D for indicated times. Total RNA was isolated and subjected to quantitative real-time PCR analysis for MMP2 mRNA levels; GAPDH was used as internal control. ∗p = 0.0082 (significant difference) (G) Cell lysates extracted from indicated cells were evaluated by western blots to determine MMP2 protein expression; β-actin was loading control. (H and I) U5637(SNHG1/Vector) cells versus U5637(SNHG1/miR-34a) cells were evaluated in a Transwell invasion assay (H); invasion rate between them was normalized to insert control according to the manufacturer’s instructions (I). Results shown are means ± SD from at least triplicate experiments. ∗p = 0.0074 (significant difference).
Figure Legend Snippet: miR-34a Downregulation Was Crucial for SNHG1-Mediated MMP2 mRNA Stabilization and Invasion in Basal MIBC Cells (A) An MMP2 mRNA 3′ UTR luciferase reporter was transiently transfected into the indicated cells, and the luciferase activity of each transfectant was evaluated. Luciferase activity is presented as relative to vector transfectant with normalization to internal transfection control pRL-TK. ∗p = 0.0083 (significant difference). (B) Quantitative real-time PCR was used to determine expression of miRNA in indicated cells. ∗p = 0.0091 (significant difference). (C) Schematic of construction of the MMP2 mRNA 3′ UTR luciferase reporter and its mutants were aligned with miR-34a. (D) WT and mutant MMP2 mRNA 3′ UTR luciferase reporters were transiently co-transfected with pRL-TK into indicated cells. Luciferase activity of each transfectant was evaluated; results are presented as relative MMP2 mRNA 3′ UTR activity. ∗p = 0.0063 (significant difference). (E) miR-34a constitutively expressed plasmids were stably transfected into U5637(SNHG1) cells. Stable transfectants were identified by real-time PCR. ∗p = 0.0065 (significant difference). (F) U5637(SNHG1/miR-34a) cells and their scramble vector transfectant were seeded into 6-well plates. After synchronization, cells were treated with Act D for indicated times. Total RNA was isolated and subjected to quantitative real-time PCR analysis for MMP2 mRNA levels; GAPDH was used as internal control. ∗p = 0.0082 (significant difference) (G) Cell lysates extracted from indicated cells were evaluated by western blots to determine MMP2 protein expression; β-actin was loading control. (H and I) U5637(SNHG1/Vector) cells versus U5637(SNHG1/miR-34a) cells were evaluated in a Transwell invasion assay (H); invasion rate between them was normalized to insert control according to the manufacturer’s instructions (I). Results shown are means ± SD from at least triplicate experiments. ∗p = 0.0074 (significant difference).

Techniques Used: Luciferase, Transfection, Activity Assay, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing, Mutagenesis, Stable Transfection, Isolation, Western Blot, Transwell Invasion Assay

14) Product Images from "MicroRNA-141 inhibits migration of gastric cancer by targeting zinc finger E-box-binding homeobox 2"

Article Title: MicroRNA-141 inhibits migration of gastric cancer by targeting zinc finger E-box-binding homeobox 2

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2015.3789

Zinc finger E-box-binding homeobox 2 (ZEB2) is the target of microRNA (miR)-141. (A) Schematic diagram of the putative miR-141-binding sites in the ZEB2 3′-untranslated region (UTR) region, as detected by TargetScan. ZEB2-mut indicates the ZEB2 3′-UTR with mutation in miR-141-binding sites. (B and C) Regulation of luciferase activity by ZEB2 3′-UTR is dependent on miR-141. (B) HGC-27 and (C) SGC-7901 human gastric cancer cells were transfected with the pRL-TK containing a Renilla luciferase gene, and the indicated vectors or precursors. Bars indicate the Firefly luciferase activities normalized to Renilla luciferase activities of the cotransfected pRL-TKvector. Each experiment was repeated at least three times, and each sample was assayed in triplicate. Data is expressed as the mean ± standard error from three independent experiments. * P
Figure Legend Snippet: Zinc finger E-box-binding homeobox 2 (ZEB2) is the target of microRNA (miR)-141. (A) Schematic diagram of the putative miR-141-binding sites in the ZEB2 3′-untranslated region (UTR) region, as detected by TargetScan. ZEB2-mut indicates the ZEB2 3′-UTR with mutation in miR-141-binding sites. (B and C) Regulation of luciferase activity by ZEB2 3′-UTR is dependent on miR-141. (B) HGC-27 and (C) SGC-7901 human gastric cancer cells were transfected with the pRL-TK containing a Renilla luciferase gene, and the indicated vectors or precursors. Bars indicate the Firefly luciferase activities normalized to Renilla luciferase activities of the cotransfected pRL-TKvector. Each experiment was repeated at least three times, and each sample was assayed in triplicate. Data is expressed as the mean ± standard error from three independent experiments. * P

Techniques Used: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Transfection

15) Product Images from "Targeting a pre-mRNA structure with bipartite antisense molecules modulates tau alternative splicing"

Article Title: Targeting a pre-mRNA structure with bipartite antisense molecules modulates tau alternative splicing

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks710

Bipartite RNA ASO inhibits exon 10 splicing from a tau minigene expressed in SK-N-SH cells and from the endogenous tau pre-mRNA in HEK-293 cells, as quantified by real-time PCR. 10 nM 9-1A-10 RNA or E10B RNA caused a significant reduction in exon 10 splicing from the wild-type minigene ( A ), and from the DDPAC minigene ( B ), with a concomitant increase in the 3R isoform compared with 10 nM scrambled control RNA. ( C ) The inhibition of exon 10 splicing by 9-1A-10 RNA was concentration-dependent, with an IC 50 of 5.4 nM. ( D ) 9-1A-10 RNA ASO does not alter total tau expression from the tau minigene in SK-N-SH cells. Total tau expression from the minigene in transfected cells was normalized to the expression of pRL-TK, which was used as a transfection control. ( E ) Transfection of HEK-293 cells with 1 µM 9-1A-10 or E10B antisense RNA significantly inhibits exon 10 splicing from the endogenous tau pre-mRNA in comparison with transfection with a scrambled control. ( F ) Total endogenous tau is unaltered in cells transfected with 1 µM 9-1A-10, E10B or scrambled RNAs. Expression of total tau was normalized to expression of GAPDH.
Figure Legend Snippet: Bipartite RNA ASO inhibits exon 10 splicing from a tau minigene expressed in SK-N-SH cells and from the endogenous tau pre-mRNA in HEK-293 cells, as quantified by real-time PCR. 10 nM 9-1A-10 RNA or E10B RNA caused a significant reduction in exon 10 splicing from the wild-type minigene ( A ), and from the DDPAC minigene ( B ), with a concomitant increase in the 3R isoform compared with 10 nM scrambled control RNA. ( C ) The inhibition of exon 10 splicing by 9-1A-10 RNA was concentration-dependent, with an IC 50 of 5.4 nM. ( D ) 9-1A-10 RNA ASO does not alter total tau expression from the tau minigene in SK-N-SH cells. Total tau expression from the minigene in transfected cells was normalized to the expression of pRL-TK, which was used as a transfection control. ( E ) Transfection of HEK-293 cells with 1 µM 9-1A-10 or E10B antisense RNA significantly inhibits exon 10 splicing from the endogenous tau pre-mRNA in comparison with transfection with a scrambled control. ( F ) Total endogenous tau is unaltered in cells transfected with 1 µM 9-1A-10, E10B or scrambled RNAs. Expression of total tau was normalized to expression of GAPDH.

Techniques Used: Allele-specific Oligonucleotide, Real-time Polymerase Chain Reaction, Inhibition, Concentration Assay, Expressing, Transfection

16) Product Images from "Dominant ER Stress–Inducing WFS1 Mutations Underlie a Genetic Syndrome of Neonatal/Infancy-Onset Diabetes, Congenital Sensorineural Deafness, and Congenital Cataracts"

Article Title: Dominant ER Stress–Inducing WFS1 Mutations Underlie a Genetic Syndrome of Neonatal/Infancy-Onset Diabetes, Congenital Sensorineural Deafness, and Congenital Cataracts

Journal: Diabetes

doi: 10.2337/db16-1296

Luciferase reporter assays in HeLa cells transfected with the ERSE reporter together with control (pcDNA) (EV), wild-type WFS1 (WT), mutant c.2171C > T p.Pro724Leu (P724L), mutant c.937C > T p.His313Tyr (p.H313Y), mutant c.2489A > C p.Glu830Ala (p.E830A), or mutant c.2425G > A p.Glu809Lys (p.E809K) expression plasmid. Cells were untreated (UT) or treated with TG (100 nmol/L) for 8 h. Relative intensity of luciferase (Promega Dual-Luciferase Reporter Assay System) was then measured ( n = 3; dashed lines represent mean). Transfections were normalized with the pRL-TK vector (Promega) as an internal control. Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett test: * P
Figure Legend Snippet: Luciferase reporter assays in HeLa cells transfected with the ERSE reporter together with control (pcDNA) (EV), wild-type WFS1 (WT), mutant c.2171C > T p.Pro724Leu (P724L), mutant c.937C > T p.His313Tyr (p.H313Y), mutant c.2489A > C p.Glu830Ala (p.E830A), or mutant c.2425G > A p.Glu809Lys (p.E809K) expression plasmid. Cells were untreated (UT) or treated with TG (100 nmol/L) for 8 h. Relative intensity of luciferase (Promega Dual-Luciferase Reporter Assay System) was then measured ( n = 3; dashed lines represent mean). Transfections were normalized with the pRL-TK vector (Promega) as an internal control. Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett test: * P

Techniques Used: Luciferase, Transfection, Mutagenesis, Expressing, Plasmid Preparation, Reporter Assay

17) Product Images from "Grass Carp Laboratory of Genetics and Physiology 2 Serves As a Negative Regulator in Retinoic Acid-Inducible Gene I- and Melanoma Differentiation-Associated Gene 5-Mediated Antiviral Signaling in Resting State and Early Stage of Grass Carp Reovirus Infection"

Article Title: Grass Carp Laboratory of Genetics and Physiology 2 Serves As a Negative Regulator in Retinoic Acid-Inducible Gene I- and Melanoma Differentiation-Associated Gene 5-Mediated Antiviral Signaling in Resting State and Early Stage of Grass Carp Reovirus Infection

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00352

Laboratory of genetics and physiology 2 (LGP2) interacts with melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I) independent of grass carp reovirus (GCRV) infection . (A,B) LGP2 overexpression inhibits RIG-I and MDA5 promoter activities. Fathead minnow (FHM) cells were transiently transfected with 300 ng of LGP2-Flag overexpression plasmid or empty vector, 30 ng of pRL-TK, and 300 ng of report vector (RIG-Ipro-luc or MDA5pro-luc) for 16 h, and then the cells were infected with GCRV or uninfected. Dual-luciferase report assays were conducted at 24 h after GCRV infection. Error bars indicate SD ( n = 4). Asterisks indicate significant differences from control (*0.01
Figure Legend Snippet: Laboratory of genetics and physiology 2 (LGP2) interacts with melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I) independent of grass carp reovirus (GCRV) infection . (A,B) LGP2 overexpression inhibits RIG-I and MDA5 promoter activities. Fathead minnow (FHM) cells were transiently transfected with 300 ng of LGP2-Flag overexpression plasmid or empty vector, 30 ng of pRL-TK, and 300 ng of report vector (RIG-Ipro-luc or MDA5pro-luc) for 16 h, and then the cells were infected with GCRV or uninfected. Dual-luciferase report assays were conducted at 24 h after GCRV infection. Error bars indicate SD ( n = 4). Asterisks indicate significant differences from control (*0.01

Techniques Used: Infection, Over Expression, Transfection, Plasmid Preparation, Luciferase

Laboratory of genetics and physiology 2 (LGP2) inhibits downstream signaling of retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) . (A,B) LGP2 inhibits RIG-I-mediated IFN-β promoter stimulator 1 (IPS-1) and mediator of IRF3 activation (MITA) promoter activities. Fathead minnow (FHM) cells were transiently transfected with 200 ng of LGP2-Flag or empty vector, 200 ng of RIG-I expression plasmid, 30 ng of pRL-TK plus 200 ng of IPS-1pro-luc or MITApro-luc for 16 h and then infected with grass carp reovirus (GCRV). Luciferase activities were conducted at 24 h after GCRV infection. (C,D) LGP2 inhibits MDA5-mediated IPS-1, but not MITA promoter activity. FHM cells were transiently transfected with 200 ng of LGP2-Flag or empty vector, 200 ng of MDA5 expression plasmids, 30 ng of pRL-TK plus 200 ng of IPS-1pro-luc or MITApro-luc in 24-well plates. At 16 h post-transfection, the cells were infected with GCRV for 24 h and then subjected to luciferase activities analysis. (E) Upper: schematic representations of full-length RIG-I and the three domains constructed in the present study. Below: LGP2 interacts with RIG-I-Helicase, RIG-I-RD, but not RIG-I-CARDs domain. FHM cells were cotransfected with 4 µg LGP2-Flag and 4 µg RIG-I-CARD-HA or RIG-I-Helicase-HA or RIG-I-RD-HA for 24 h in 10 cm 2 dishes. Co-IP was performed using anti-HA antibody (Ab), and mouse IgG was used as control. IPs were analyzed by IBs with anti-HA and anti-Flag, respectively. Expression of LGP2-Flag (input) was examined with anti-Flag. (F) Upper: full-length MDA5 and its domain structures. Below: LGP2 interacts with MDA5-CARDs, MDA5-Helicase, and MDA5-RD. FHM cells were transfected with the indicated plasmids (4 µg each). Twenty-four hours later, cells were lysed, Co-IP and IB analyses were performed with the indicated Abs. (G) LGP2 inhibits RIG-I-CARDs-mediated MITA, but not IPS-1 promoter activity. FHM cells were transfected with 200 ng of LGP2-Flag or empty vector, 200 ng of RIG-I-CARD-HA expression plasmid, 30 ng of pRL-TK plus 200 ng of IPS-1pro-luc or MITApro-luc. Luciferase assays were performed at 24 h post-transfection. (H) LGP2 inhibits MDA5-CARDs-mediated IPS-1 and MITA promoter activities. FHM cells were transfected with 200 ng of LGP2-Flag or empty vector, 200 ng of MDA5-CARD-HA expression plasmid, 30 ng of pRL-TK plus 200 ng of IPS-1pro-luc or MITApro-luc. Luciferase assays were performed at 24 h post-transfection. Error bars indicate SD ( n = 4). Asterisks indicate significant differences from control (**0.001
Figure Legend Snippet: Laboratory of genetics and physiology 2 (LGP2) inhibits downstream signaling of retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) . (A,B) LGP2 inhibits RIG-I-mediated IFN-β promoter stimulator 1 (IPS-1) and mediator of IRF3 activation (MITA) promoter activities. Fathead minnow (FHM) cells were transiently transfected with 200 ng of LGP2-Flag or empty vector, 200 ng of RIG-I expression plasmid, 30 ng of pRL-TK plus 200 ng of IPS-1pro-luc or MITApro-luc for 16 h and then infected with grass carp reovirus (GCRV). Luciferase activities were conducted at 24 h after GCRV infection. (C,D) LGP2 inhibits MDA5-mediated IPS-1, but not MITA promoter activity. FHM cells were transiently transfected with 200 ng of LGP2-Flag or empty vector, 200 ng of MDA5 expression plasmids, 30 ng of pRL-TK plus 200 ng of IPS-1pro-luc or MITApro-luc in 24-well plates. At 16 h post-transfection, the cells were infected with GCRV for 24 h and then subjected to luciferase activities analysis. (E) Upper: schematic representations of full-length RIG-I and the three domains constructed in the present study. Below: LGP2 interacts with RIG-I-Helicase, RIG-I-RD, but not RIG-I-CARDs domain. FHM cells were cotransfected with 4 µg LGP2-Flag and 4 µg RIG-I-CARD-HA or RIG-I-Helicase-HA or RIG-I-RD-HA for 24 h in 10 cm 2 dishes. Co-IP was performed using anti-HA antibody (Ab), and mouse IgG was used as control. IPs were analyzed by IBs with anti-HA and anti-Flag, respectively. Expression of LGP2-Flag (input) was examined with anti-Flag. (F) Upper: full-length MDA5 and its domain structures. Below: LGP2 interacts with MDA5-CARDs, MDA5-Helicase, and MDA5-RD. FHM cells were transfected with the indicated plasmids (4 µg each). Twenty-four hours later, cells were lysed, Co-IP and IB analyses were performed with the indicated Abs. (G) LGP2 inhibits RIG-I-CARDs-mediated MITA, but not IPS-1 promoter activity. FHM cells were transfected with 200 ng of LGP2-Flag or empty vector, 200 ng of RIG-I-CARD-HA expression plasmid, 30 ng of pRL-TK plus 200 ng of IPS-1pro-luc or MITApro-luc. Luciferase assays were performed at 24 h post-transfection. (H) LGP2 inhibits MDA5-CARDs-mediated IPS-1 and MITA promoter activities. FHM cells were transfected with 200 ng of LGP2-Flag or empty vector, 200 ng of MDA5-CARD-HA expression plasmid, 30 ng of pRL-TK plus 200 ng of IPS-1pro-luc or MITApro-luc. Luciferase assays were performed at 24 h post-transfection. Error bars indicate SD ( n = 4). Asterisks indicate significant differences from control (**0.001

Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Expressing, Infection, Luciferase, Activity Assay, Construct, Co-Immunoprecipitation Assay

Identification of laboratory of genetics and physiology 2 (LGP2) as an inhibitor in IFNs and NF-κBs activation . (A,B) LGP2 overexpression suppresses the promoter activities of IFNs and NF-κBs. Fathead minnow (FHM) cells were cotransfected with 300 ng of LGP2-Flag overexpression plasmid, 30 ng of pRL-TK, and 300 ng of IFN1pro-luc, IFN2pro-luc, IFN3pro-luc, IFN4pro-luc, IFNγ1pro-luc, IFNγ2pro-luc, NF-κB1pro-luc, NF-κB2pro-luc in 24-well plates. Control was transfected with 300 ng of empty vector (pCMV-CMV-GFP), same amount of the corresponding report vectors, and pRL-TK. At 16 h post-transfection, the cells were infected with grass carp reovirus (GCRV) or uninfected. Dual-luciferase report assays were conducted at 12 h (IFN2, IFN4, IFNγ2) or 24 h (IFN1, IFN3, IFNγ1, NF-κB1, NF-κB2) after GCRV infection. Time-matched mocks were treated with PBS. Error bars indicate SD ( n = 4). Asterisks indicate significant differences from control (**0.001
Figure Legend Snippet: Identification of laboratory of genetics and physiology 2 (LGP2) as an inhibitor in IFNs and NF-κBs activation . (A,B) LGP2 overexpression suppresses the promoter activities of IFNs and NF-κBs. Fathead minnow (FHM) cells were cotransfected with 300 ng of LGP2-Flag overexpression plasmid, 30 ng of pRL-TK, and 300 ng of IFN1pro-luc, IFN2pro-luc, IFN3pro-luc, IFN4pro-luc, IFNγ1pro-luc, IFNγ2pro-luc, NF-κB1pro-luc, NF-κB2pro-luc in 24-well plates. Control was transfected with 300 ng of empty vector (pCMV-CMV-GFP), same amount of the corresponding report vectors, and pRL-TK. At 16 h post-transfection, the cells were infected with grass carp reovirus (GCRV) or uninfected. Dual-luciferase report assays were conducted at 12 h (IFN2, IFN4, IFNγ2) or 24 h (IFN1, IFN3, IFNγ1, NF-κB1, NF-κB2) after GCRV infection. Time-matched mocks were treated with PBS. Error bars indicate SD ( n = 4). Asterisks indicate significant differences from control (**0.001

Techniques Used: Activation Assay, Over Expression, Plasmid Preparation, Transfection, Infection, Luciferase

18) Product Images from "IGFBP3, a Transcriptional Target of Homeobox D10, Is Correlated with the Prognosis of Gastric Cancer"

Article Title: IGFBP3, a Transcriptional Target of Homeobox D10, Is Correlated with the Prognosis of Gastric Cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0081423

Identification of the regulatory regions of IGFBP3 which are responsible by HoxD10 in BGC823 cells. ( A ) BGC823 cells were transfected with pcDNA3.1 empty vector or pcDNA3.1-HoxD10, pGL3-promoter vector or pGL3-HBSI(II)-promoter and pRL-TK vector. ( B ) Different oligonucleotides which contain wild or point mutant sequences of HBS3, HBS4 and HBS5 were cloned into pGL3-HBS-promoter. Relative firefly activity was expressed normalized to renilla activity in pRL-TK vector. All experiments were performed in triplicate. ** indicates of p
Figure Legend Snippet: Identification of the regulatory regions of IGFBP3 which are responsible by HoxD10 in BGC823 cells. ( A ) BGC823 cells were transfected with pcDNA3.1 empty vector or pcDNA3.1-HoxD10, pGL3-promoter vector or pGL3-HBSI(II)-promoter and pRL-TK vector. ( B ) Different oligonucleotides which contain wild or point mutant sequences of HBS3, HBS4 and HBS5 were cloned into pGL3-HBS-promoter. Relative firefly activity was expressed normalized to renilla activity in pRL-TK vector. All experiments were performed in triplicate. ** indicates of p

Techniques Used: Transfection, Plasmid Preparation, Mutagenesis, Clone Assay, Activity Assay

The expression of IGFBP3 is regulated by HoxD10 in human gastric cancer cells. The expression of IGFBP3 was detected by conventional RT-PCR ( A , C ) and Western blotting ( B , D ) after transfected with pcDNA3.1 empty vector or pcDNA3.1-HoxD10 in BGC823 and SGC7901 cells, and negative control siRNA (Ne-Si) or two of HoxD10 siRNA (H-Si1 and H-Si2) in HGC27 cells. GAPDH was used as internal control. Densitometry values are expressed as fold change compared with pCDNA3.1 vector or negative control siRNA values normalized to 1. ( E ) BGC823 and SGC7901 were co-transfected with pcDNA3.1 empty vector or pcDNA3.1-HoxD10, pGL3-basic vector or pGL3-(pIGFBP3)-basic and pRL-TK vector. Relative firefly activity was expressed normalized to renilla activity in pRL-TK vector. All experiments were performed in triplicate. ** indicates of p
Figure Legend Snippet: The expression of IGFBP3 is regulated by HoxD10 in human gastric cancer cells. The expression of IGFBP3 was detected by conventional RT-PCR ( A , C ) and Western blotting ( B , D ) after transfected with pcDNA3.1 empty vector or pcDNA3.1-HoxD10 in BGC823 and SGC7901 cells, and negative control siRNA (Ne-Si) or two of HoxD10 siRNA (H-Si1 and H-Si2) in HGC27 cells. GAPDH was used as internal control. Densitometry values are expressed as fold change compared with pCDNA3.1 vector or negative control siRNA values normalized to 1. ( E ) BGC823 and SGC7901 were co-transfected with pcDNA3.1 empty vector or pcDNA3.1-HoxD10, pGL3-basic vector or pGL3-(pIGFBP3)-basic and pRL-TK vector. Relative firefly activity was expressed normalized to renilla activity in pRL-TK vector. All experiments were performed in triplicate. ** indicates of p

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, Negative Control, Activity Assay

19) Product Images from "The unfolded protein response induced by Tembusu virus infection"

Article Title: The unfolded protein response induced by Tembusu virus infection

Journal: BMC Veterinary Research

doi: 10.1186/s12917-019-1781-4

Analysis of the ATF6 pathway during TMUV infection. a , Expression of ATF6 after TMUV infection was examined by western blotting using an antibody specific for full-length ATF6. Tubulin was used as an internal control. The intensities of bands were determined using IMAGE J software, and the data represent ratios of TMUV-infected cells to mock-infected cells at the indicated time points. b , Activation of the ATF6 pathway was monitored by a dual luciferase reporter gene assay. BHK-21 cells were transfected with pGM-ATF6-Lu or pGM-ERSE-Lu. Firefly luciferase activity was normalized based on Renilla luciferase activity in cells cotransfected with pRL-TK. After 48 h of transfection, the cells were mock infected or infected with TMUV. Cells treated with 1 μM tunicamycin for 12 h or 2.5 mM dithiothreitol (DTT) for 7 h were used as a positive control. Cells were collected at the indicated time points and assayed for firefly and Renilla luciferase activities. The values represent the means±SD of results from three independent experiments. c , Expression of chaperones induced by TMUV infection. The Y axis shows the fold change of target gene expression in TMUV-infected cells, as determined by the comparative CT method. The data are expressed as the means±SD of results from three independent experiments. * p
Figure Legend Snippet: Analysis of the ATF6 pathway during TMUV infection. a , Expression of ATF6 after TMUV infection was examined by western blotting using an antibody specific for full-length ATF6. Tubulin was used as an internal control. The intensities of bands were determined using IMAGE J software, and the data represent ratios of TMUV-infected cells to mock-infected cells at the indicated time points. b , Activation of the ATF6 pathway was monitored by a dual luciferase reporter gene assay. BHK-21 cells were transfected with pGM-ATF6-Lu or pGM-ERSE-Lu. Firefly luciferase activity was normalized based on Renilla luciferase activity in cells cotransfected with pRL-TK. After 48 h of transfection, the cells were mock infected or infected with TMUV. Cells treated with 1 μM tunicamycin for 12 h or 2.5 mM dithiothreitol (DTT) for 7 h were used as a positive control. Cells were collected at the indicated time points and assayed for firefly and Renilla luciferase activities. The values represent the means±SD of results from three independent experiments. c , Expression of chaperones induced by TMUV infection. The Y axis shows the fold change of target gene expression in TMUV-infected cells, as determined by the comparative CT method. The data are expressed as the means±SD of results from three independent experiments. * p

Techniques Used: Infection, Expressing, Western Blot, Software, Activation Assay, Luciferase, Reporter Gene Assay, Transfection, Activity Assay, Positive Control

20) Product Images from "Zebrafish TRIM25 Promotes Innate Immune Response to RGNNV Infection by Targeting 2CARD and RD Regions of RIG-I for K63-Linked Ubiquitination"

Article Title: Zebrafish TRIM25 Promotes Innate Immune Response to RGNNV Infection by Targeting 2CARD and RD Regions of RIG-I for K63-Linked Ubiquitination

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.02805

zbTRIM25 potentiates RLR signaling pathway. (A,B) ZBE3 cells were transfected with pCMV-Flag-zbTRIM25 or pCMV-Flag vector for 24 h. After treated with RGNNV (A) or poly I:C (B) , the transcript levels of RIG-I, MAVS, TRAF3, IRF3, IFN 1 , and ISG15 mRNA in ZBE3 cells were analyzed. (C) HEK 293T cells were transfected with pCMV-Myc or pCMV-Myc-zbRIG-I together with pCMV-Flag or increasing amount of pCMV-Flag-zbTRIM25 as well as DrIFN1 pro-Luc and pRL-TK . Luciferase activities were measured and normalized to the amount of Renilla Luciferase activities. (D) ZBE3 cells were transfected with pCMV-Flag and increasing amount of pCMV-Flag-zbTRIM25 for 24 h, and then infected with RGNNV for 24 h. QRT-PCR analysis was performed for RDRP . (E,F) ZBE3 cells were either not transfected (Control) or transfected with 100 nM Control siRNA (NC) or 100 nM zbTRIM25 siRNA. Cells were then subcultured for 24 h and infected with RGNNV for 24 h. Cells were harvested subsequently. Levels of zbTRIM25 and RDRP mRNA were analyzed by qRT-PCR and normalized with 18s rRNA . Data represent the mean + SD ( n = 3). Asterisks indicate significant differences between groups (* p
Figure Legend Snippet: zbTRIM25 potentiates RLR signaling pathway. (A,B) ZBE3 cells were transfected with pCMV-Flag-zbTRIM25 or pCMV-Flag vector for 24 h. After treated with RGNNV (A) or poly I:C (B) , the transcript levels of RIG-I, MAVS, TRAF3, IRF3, IFN 1 , and ISG15 mRNA in ZBE3 cells were analyzed. (C) HEK 293T cells were transfected with pCMV-Myc or pCMV-Myc-zbRIG-I together with pCMV-Flag or increasing amount of pCMV-Flag-zbTRIM25 as well as DrIFN1 pro-Luc and pRL-TK . Luciferase activities were measured and normalized to the amount of Renilla Luciferase activities. (D) ZBE3 cells were transfected with pCMV-Flag and increasing amount of pCMV-Flag-zbTRIM25 for 24 h, and then infected with RGNNV for 24 h. QRT-PCR analysis was performed for RDRP . (E,F) ZBE3 cells were either not transfected (Control) or transfected with 100 nM Control siRNA (NC) or 100 nM zbTRIM25 siRNA. Cells were then subcultured for 24 h and infected with RGNNV for 24 h. Cells were harvested subsequently. Levels of zbTRIM25 and RDRP mRNA were analyzed by qRT-PCR and normalized with 18s rRNA . Data represent the mean + SD ( n = 3). Asterisks indicate significant differences between groups (* p

Techniques Used: Transfection, Plasmid Preparation, Luciferase, Infection, Quantitative RT-PCR

zbTRIM25-mediated K63 ubiquitination in 2CARD and RD regions of zbRIG-I is important for IFN induction. (A) HEK 293T cells were transfected with pEGFP-zbRIG-I, pEGFP-zbRIG-I-2CARD , and pEGFP-zbRIG-I-RD as well as DrIFN1 pro-Luc and pRL-TK vectors. (B,C) HEK 293T cells were transfected with pEGFP-zbRIG-I-2CARD or pEGFP-zbRIG-I-RD together with HA-K63Ub and increasing amount of pCMV-Flag-zbTRIM25 as well as DrIFN1 pro-Luc and pRL-TK plasmids. Luciferase activities were measured and normalized to the amount of Renilla Luciferase activities. Data represent the mean + SD ( n = 3). Asterisks indicate significant differences between groups (* p
Figure Legend Snippet: zbTRIM25-mediated K63 ubiquitination in 2CARD and RD regions of zbRIG-I is important for IFN induction. (A) HEK 293T cells were transfected with pEGFP-zbRIG-I, pEGFP-zbRIG-I-2CARD , and pEGFP-zbRIG-I-RD as well as DrIFN1 pro-Luc and pRL-TK vectors. (B,C) HEK 293T cells were transfected with pEGFP-zbRIG-I-2CARD or pEGFP-zbRIG-I-RD together with HA-K63Ub and increasing amount of pCMV-Flag-zbTRIM25 as well as DrIFN1 pro-Luc and pRL-TK plasmids. Luciferase activities were measured and normalized to the amount of Renilla Luciferase activities. Data represent the mean + SD ( n = 3). Asterisks indicate significant differences between groups (* p

Techniques Used: Transfection, Luciferase

21) Product Images from "Indoxyl sulfate induces intestinal barrier injury through IRF1-DRP1 axis-mediated mitophagy impairment"

Article Title: Indoxyl sulfate induces intestinal barrier injury through IRF1-DRP1 axis-mediated mitophagy impairment

Journal: Theranostics

doi: 10.7150/thno.45455

IRF1 suppresses DRP1 transcription through directly binding to its promoter region. (A) Caco2 cells were co-transfected with pRL-TK vector and pGL3-basic or recombinant plasmids containing various DRP1 promoter regions, in combination with pControl or pIRF1. Then, cells were harvested for dual-luciferase reporter assay. Firefly luciferase activity was normalized against Renilla activity. (B, C) Cells were co-transfected with pRL-TK and pGL3-basic, pGL3-DPR1-P4, pGL3-DPR1-M4, pGL3-DPR1-P3 or pGL3-DPR1-M3 (the mutated IRF1 binding sites were underlined) using Lipofectamine 2000, and treated with control or IS for another 24 hours for dual-luciferase reporter assay. (D) Cells were treated with control or IS for 24 hours and collected for ChIP assay. DNA was amplified using PCR primers (-584 ~ -401 and -1136 ~ -1020). (E) qPCR analysis of IRF1 antibody-immunoprecipitated DNA in (D) . (F, G) Cells were transfected with siAhR or siControl and treated with control or IS for another 24 hours. Cells were harvested for Western blot analysis to detect AhR and IRF1 expression (F) and for immunofluorescence confocal microscopy to evaluate the cellular localization of AhR (G) . Scale bar, 10 μm. The gray scale of bands was quantified using ImageJ software. Data are shown as mean ± SEM and were analyzed by two-tailed unpaired Student's t test ( A-C, E ) or one-way ANOVA ( F ). n=3. ns: no significance. * P
Figure Legend Snippet: IRF1 suppresses DRP1 transcription through directly binding to its promoter region. (A) Caco2 cells were co-transfected with pRL-TK vector and pGL3-basic or recombinant plasmids containing various DRP1 promoter regions, in combination with pControl or pIRF1. Then, cells were harvested for dual-luciferase reporter assay. Firefly luciferase activity was normalized against Renilla activity. (B, C) Cells were co-transfected with pRL-TK and pGL3-basic, pGL3-DPR1-P4, pGL3-DPR1-M4, pGL3-DPR1-P3 or pGL3-DPR1-M3 (the mutated IRF1 binding sites were underlined) using Lipofectamine 2000, and treated with control or IS for another 24 hours for dual-luciferase reporter assay. (D) Cells were treated with control or IS for 24 hours and collected for ChIP assay. DNA was amplified using PCR primers (-584 ~ -401 and -1136 ~ -1020). (E) qPCR analysis of IRF1 antibody-immunoprecipitated DNA in (D) . (F, G) Cells were transfected with siAhR or siControl and treated with control or IS for another 24 hours. Cells were harvested for Western blot analysis to detect AhR and IRF1 expression (F) and for immunofluorescence confocal microscopy to evaluate the cellular localization of AhR (G) . Scale bar, 10 μm. The gray scale of bands was quantified using ImageJ software. Data are shown as mean ± SEM and were analyzed by two-tailed unpaired Student's t test ( A-C, E ) or one-way ANOVA ( F ). n=3. ns: no significance. * P

Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Recombinant, Luciferase, Reporter Assay, Activity Assay, Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Immunoprecipitation, Western Blot, Expressing, Immunofluorescence, Confocal Microscopy, Software, Two Tailed Test

22) Product Images from "Regulation of Human Formyl Peptide Receptor 1 Synthesis: Role of Single Nucleotide Polymorphisms, Transcription Factors, and Inflammatory Mediators"

Article Title: Regulation of Human Formyl Peptide Receptor 1 Synthesis: Role of Single Nucleotide Polymorphisms, Transcription Factors, and Inflammatory Mediators

Journal: PLoS ONE

doi: 10.1371/journal.pone.0028712

Location of putative transcription factor binding sites on FPR1 promoter. A. Sequence analysis using PROMO3 software identified certain transcription factors commonly expressed in myeloid cells as putative regulators of FPR1 transcription. The numbers indicate the first nucleotide of the various promoter constructs in relation to the transcriptional start site (TSS). The −56 and −54 SNPs are underlined and the various mutations of putative transcription factor binding sites are in bold. B. Site-directed mutagenesis of the putative PU.1 and STAT4 binding sites resulted in a significant decrease in firefly luciferase activity. U937 cells were co-transfected with the indicated wild-type and promoter mutant constructs and pRL-TK to normalize for transfection efficiency. Data show the mean ratios from three experiments ± S.E.M. One-way analysis of variance showed that differences in luciferase activity among the constructs were significant ( p value
Figure Legend Snippet: Location of putative transcription factor binding sites on FPR1 promoter. A. Sequence analysis using PROMO3 software identified certain transcription factors commonly expressed in myeloid cells as putative regulators of FPR1 transcription. The numbers indicate the first nucleotide of the various promoter constructs in relation to the transcriptional start site (TSS). The −56 and −54 SNPs are underlined and the various mutations of putative transcription factor binding sites are in bold. B. Site-directed mutagenesis of the putative PU.1 and STAT4 binding sites resulted in a significant decrease in firefly luciferase activity. U937 cells were co-transfected with the indicated wild-type and promoter mutant constructs and pRL-TK to normalize for transfection efficiency. Data show the mean ratios from three experiments ± S.E.M. One-way analysis of variance showed that differences in luciferase activity among the constructs were significant ( p value

Techniques Used: Binding Assay, Sequencing, Software, Construct, Mutagenesis, Luciferase, Activity Assay, Transfection

Identification of the minimal promoter region of FPR1 . Serial deletion fragments of the FPR1 promoter were generated by PCR and cloned upstream from the luciferase reporter gene in the pGL3 Basic vector. 10 µg of pGL3-Control vector containing the SV40 promoter was used as positive control and 30 µg of pGL3-Basic lacking a promoter was used to measure background luminescence. The amount of pGL3-Basic- FPR1 promoter plasmids in all experiments was 30 µg. U937 cells were co-electroporated with the firefly luciferase plasmids and 300 ng of pRL-TK as a transfection standard. Results show the mean ratios of firefly to Renilla luciferase 24 hours post-transfection from 6–19 separate experiments ± S.E.M. Unpaired t test demonstrated that the luciferase activity of the −72/41 construct was significantly lower than the activity of the −88/41 construct, ** p -value
Figure Legend Snippet: Identification of the minimal promoter region of FPR1 . Serial deletion fragments of the FPR1 promoter were generated by PCR and cloned upstream from the luciferase reporter gene in the pGL3 Basic vector. 10 µg of pGL3-Control vector containing the SV40 promoter was used as positive control and 30 µg of pGL3-Basic lacking a promoter was used to measure background luminescence. The amount of pGL3-Basic- FPR1 promoter plasmids in all experiments was 30 µg. U937 cells were co-electroporated with the firefly luciferase plasmids and 300 ng of pRL-TK as a transfection standard. Results show the mean ratios of firefly to Renilla luciferase 24 hours post-transfection from 6–19 separate experiments ± S.E.M. Unpaired t test demonstrated that the luciferase activity of the −72/41 construct was significantly lower than the activity of the −88/41 construct, ** p -value

Techniques Used: Generated, Polymerase Chain Reaction, Clone Assay, Luciferase, Plasmid Preparation, Positive Control, Transfection, Activity Assay, Construct

Four FPR1 variants show similar expression levels. FPR1 haplotypes 8A, 11A, 12D and 16A were expressed as fusion proteins with firefly luciferase in U937 cells. Cells were electroporated with various amounts of the firefly luciferase reporter plasmid (as shown) and 300 ng pRL-TK Renilla luciferase control reporter plasmid. 24 h post-transfection cell extracts were analyzed using the Promega dual luciferase assay kit. The graphs show the mean ratios of firefly and Renilla luciferase from five separate experiments ± S.E.M. One-way analysis of variance showed no statistical differences between the haplotypes.
Figure Legend Snippet: Four FPR1 variants show similar expression levels. FPR1 haplotypes 8A, 11A, 12D and 16A were expressed as fusion proteins with firefly luciferase in U937 cells. Cells were electroporated with various amounts of the firefly luciferase reporter plasmid (as shown) and 300 ng pRL-TK Renilla luciferase control reporter plasmid. 24 h post-transfection cell extracts were analyzed using the Promega dual luciferase assay kit. The graphs show the mean ratios of firefly and Renilla luciferase from five separate experiments ± S.E.M. One-way analysis of variance showed no statistical differences between the haplotypes.

Techniques Used: Expressing, Luciferase, Plasmid Preparation, Transfection

23) Product Images from "Follicular Stimulating Hormone Accelerates Atherogenesis by Increasing Endothelial VCAM-1 Expression"

Article Title: Follicular Stimulating Hormone Accelerates Atherogenesis by Increasing Endothelial VCAM-1 Expression

Journal: Theranostics

doi: 10.7150/thno.21216

FSH activated NF-κB signaling through IKK/IkBα pathway. (A). HUVECs were treated with control vehicle (CON), FSH (50 mIU/mL) or TNF-α (0.1 ng/mL) for 24 h in the presence or absence of 17β-estradiol (E2, 10 nM). Subcellular localization of the p65 protein was assayed by immunofluorescence (red staining). Scale bar = 250 μm. (B). HUVECs were transiently transfected with 750 ng of NF-κB-luciferase reporter and 250 ng pRL-TK vector expressing renella luciferase. Subsequently, cells were treated with control vehicle (CON), FSH (50 mIU/mL) or TNF-α (0.1 ng/mL) for 24 h in the presence or absence of 17β-estradiol (E2, 10 nM). Relative luminescent units (RLU) were examined and were normalized to fold change from control. * = P
Figure Legend Snippet: FSH activated NF-κB signaling through IKK/IkBα pathway. (A). HUVECs were treated with control vehicle (CON), FSH (50 mIU/mL) or TNF-α (0.1 ng/mL) for 24 h in the presence or absence of 17β-estradiol (E2, 10 nM). Subcellular localization of the p65 protein was assayed by immunofluorescence (red staining). Scale bar = 250 μm. (B). HUVECs were transiently transfected with 750 ng of NF-κB-luciferase reporter and 250 ng pRL-TK vector expressing renella luciferase. Subsequently, cells were treated with control vehicle (CON), FSH (50 mIU/mL) or TNF-α (0.1 ng/mL) for 24 h in the presence or absence of 17β-estradiol (E2, 10 nM). Relative luminescent units (RLU) were examined and were normalized to fold change from control. * = P

Techniques Used: Immunofluorescence, Staining, Transfection, Luciferase, Plasmid Preparation, Expressing

24) Product Images from "Constitutive Expression of Murine Decay-Accelerating Factor 1 Is Controlled by the Transcription Factor Sp1 1"

Article Title: Constitutive Expression of Murine Decay-Accelerating Factor 1 Is Controlled by the Transcription Factor Sp1 1

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi:

Daf1 expression in murine fibroblasts, B cells, and macrophages. A , Constitutive Daf1 mRNA level. Total RNA was extracted from the indicated cell lines and reverse transcribed. PCR was then performed using both Daf1- and β-actin-specific primers for the indicated cycle number. PCR products were run on agarose gel and visualized with ethidium bromide. L represents DNA ladder. B , A genomic fragment corresponding to the Daf1 gene 5′ flanking region (−2408/+85 from translational start site) was cloned into the promoterless vector pGL3 encoding firefly luciferase. Either this construct or the pGL3 control vector was transiently transfected into the indicated cell line along with the transfection efficiency control plasmid pRL-TK encoding the Renilla luciferase. Luciferase activities were measured 48 h posttransfection. Data (mean ± SD) have been normalized relative to the Renilla luciferase activity.
Figure Legend Snippet: Daf1 expression in murine fibroblasts, B cells, and macrophages. A , Constitutive Daf1 mRNA level. Total RNA was extracted from the indicated cell lines and reverse transcribed. PCR was then performed using both Daf1- and β-actin-specific primers for the indicated cycle number. PCR products were run on agarose gel and visualized with ethidium bromide. L represents DNA ladder. B , A genomic fragment corresponding to the Daf1 gene 5′ flanking region (−2408/+85 from translational start site) was cloned into the promoterless vector pGL3 encoding firefly luciferase. Either this construct or the pGL3 control vector was transiently transfected into the indicated cell line along with the transfection efficiency control plasmid pRL-TK encoding the Renilla luciferase. Luciferase activities were measured 48 h posttransfection. Data (mean ± SD) have been normalized relative to the Renilla luciferase activity.

Techniques Used: Expressing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Clone Assay, Plasmid Preparation, Luciferase, Construct, Transfection, Activity Assay

Sp1 regulates Daf1 promoter activity in Drosophila Schneider SL2 cells. SL2 cells were transfected with either pGL3, p(−18/+85)Luc, p(−179/+85), p(−619/+85), or p(−2408/+85) constructs and with an increasing amount of the Sp1 containing plasmid pPacSp1 as indicated. Total amount of plasmid DNA transfected was held constant using the corresponding empty vector pPac. Renilla luciferase containing vector pRL-TK was also introduced to normalize transfection efficiency. Luciferases activities generated in the presence or absence of pPacSp1 were normalized and data (mean ± SD) are from quadruplicate transfections.
Figure Legend Snippet: Sp1 regulates Daf1 promoter activity in Drosophila Schneider SL2 cells. SL2 cells were transfected with either pGL3, p(−18/+85)Luc, p(−179/+85), p(−619/+85), or p(−2408/+85) constructs and with an increasing amount of the Sp1 containing plasmid pPacSp1 as indicated. Total amount of plasmid DNA transfected was held constant using the corresponding empty vector pPac. Renilla luciferase containing vector pRL-TK was also introduced to normalize transfection efficiency. Luciferases activities generated in the presence or absence of pPacSp1 were normalized and data (mean ± SD) are from quadruplicate transfections.

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

25) Product Images from "Caprylic acid and medium-chain triglycerides inhibit IL-8 gene transcription in Caco-2 cells: comparison with the potent histone deacetylase inhibitor trichostatin A"

Article Title: Caprylic acid and medium-chain triglycerides inhibit IL-8 gene transcription in Caco-2 cells: comparison with the potent histone deacetylase inhibitor trichostatin A

Journal: British Journal of Pharmacology

doi: 10.1038/sj.bjp.0704719

Effects of caprylic acid on activation of IL-8 promoter. Cells were transiently co-transfected with the IL-8 promoter/luciferase reporter plasmid and pRL-TK vector at a ratio of 40 : 1, and cultured with or without caprylic acid for 24 h. After stimulation with 1 ng ml −1 of IL-1β for 6 h, dual-luciferase assay was performed. The data obtained were evaluated as firefly luciferase activity/ Renilla luciferase activity (fluc/Rluc). Similar results were observed when cells were incubated with trichostatin A. Values are expressed as means±s.e.mean (* P
Figure Legend Snippet: Effects of caprylic acid on activation of IL-8 promoter. Cells were transiently co-transfected with the IL-8 promoter/luciferase reporter plasmid and pRL-TK vector at a ratio of 40 : 1, and cultured with or without caprylic acid for 24 h. After stimulation with 1 ng ml −1 of IL-1β for 6 h, dual-luciferase assay was performed. The data obtained were evaluated as firefly luciferase activity/ Renilla luciferase activity (fluc/Rluc). Similar results were observed when cells were incubated with trichostatin A. Values are expressed as means±s.e.mean (* P

Techniques Used: Activation Assay, Transfection, Luciferase, Plasmid Preparation, Cell Culture, Activity Assay, Incubation

26) Product Images from "Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation"

Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation

Journal: BioMed Research International

doi: 10.1155/2015/460598

Effect of Dex on the AQP5 promoter. EpH4 cells were treated with Dex, 4 h prior ( n = 4) or during transfection ( n = 5) of the luciferase expression pGL3-basic vector including the AQP5 promoter (FF) and the pRL-TK transfection control plasmid (R). Negative controls included the pGL3-basic vector without promoter (cFF) ( n = 2) and the peGFP-N1 vector (GFP), ( n = 1). (a) Levels of AQP5 mRNA normalized to GAPDH are shown for the different transfected EpH4 cells with or without Dex treatment. Bars represent averages and confidence intervals; alpha = 0.05. Significance was tested between samples without Dex and with Dex treatment of the same transfection type. (b) Levels of Firefly enzyme activity normalized to Renilla enzyme activity measured with the luciferase reporter assay for the same transfected cells as in panel (a). Bars represent averages and confidence intervals; alpha = 0.05. Significance was tested between controls (−Dex) and treatments (+Dex), with one or two asterisks denoting P
Figure Legend Snippet: Effect of Dex on the AQP5 promoter. EpH4 cells were treated with Dex, 4 h prior ( n = 4) or during transfection ( n = 5) of the luciferase expression pGL3-basic vector including the AQP5 promoter (FF) and the pRL-TK transfection control plasmid (R). Negative controls included the pGL3-basic vector without promoter (cFF) ( n = 2) and the peGFP-N1 vector (GFP), ( n = 1). (a) Levels of AQP5 mRNA normalized to GAPDH are shown for the different transfected EpH4 cells with or without Dex treatment. Bars represent averages and confidence intervals; alpha = 0.05. Significance was tested between samples without Dex and with Dex treatment of the same transfection type. (b) Levels of Firefly enzyme activity normalized to Renilla enzyme activity measured with the luciferase reporter assay for the same transfected cells as in panel (a). Bars represent averages and confidence intervals; alpha = 0.05. Significance was tested between controls (−Dex) and treatments (+Dex), with one or two asterisks denoting P

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

27) Product Images from "IGFBP3, a Transcriptional Target of Homeobox D10, Is Correlated with the Prognosis of Gastric Cancer"

Article Title: IGFBP3, a Transcriptional Target of Homeobox D10, Is Correlated with the Prognosis of Gastric Cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0081423

Identification of the regulatory regions of IGFBP3 which are responsible by HoxD10 in BGC823 cells. ( A ) BGC823 cells were transfected with pcDNA3.1 empty vector or pcDNA3.1-HoxD10, pGL3-promoter vector or pGL3-HBSI(II)-promoter and pRL-TK vector. ( B ) Different oligonucleotides which contain wild or point mutant sequences of HBS3, HBS4 and HBS5 were cloned into pGL3-HBS-promoter. Relative firefly activity was expressed normalized to renilla activity in pRL-TK vector. All experiments were performed in triplicate. ** indicates of p
Figure Legend Snippet: Identification of the regulatory regions of IGFBP3 which are responsible by HoxD10 in BGC823 cells. ( A ) BGC823 cells were transfected with pcDNA3.1 empty vector or pcDNA3.1-HoxD10, pGL3-promoter vector or pGL3-HBSI(II)-promoter and pRL-TK vector. ( B ) Different oligonucleotides which contain wild or point mutant sequences of HBS3, HBS4 and HBS5 were cloned into pGL3-HBS-promoter. Relative firefly activity was expressed normalized to renilla activity in pRL-TK vector. All experiments were performed in triplicate. ** indicates of p

Techniques Used: Transfection, Plasmid Preparation, Mutagenesis, Clone Assay, Activity Assay

The expression of IGFBP3 is regulated by HoxD10 in human gastric cancer cells. The expression of IGFBP3 was detected by conventional RT-PCR ( A , C ) and Western blotting ( B , D ) after transfected with pcDNA3.1 empty vector or pcDNA3.1-HoxD10 in BGC823 and SGC7901 cells, and negative control siRNA (Ne-Si) or two of HoxD10 siRNA (H-Si1 and H-Si2) in HGC27 cells. GAPDH was used as internal control. Densitometry values are expressed as fold change compared with pCDNA3.1 vector or negative control siRNA values normalized to 1. ( E ) BGC823 and SGC7901 were co-transfected with pcDNA3.1 empty vector or pcDNA3.1-HoxD10, pGL3-basic vector or pGL3-(pIGFBP3)-basic and pRL-TK vector. Relative firefly activity was expressed normalized to renilla activity in pRL-TK vector. All experiments were performed in triplicate. ** indicates of p
Figure Legend Snippet: The expression of IGFBP3 is regulated by HoxD10 in human gastric cancer cells. The expression of IGFBP3 was detected by conventional RT-PCR ( A , C ) and Western blotting ( B , D ) after transfected with pcDNA3.1 empty vector or pcDNA3.1-HoxD10 in BGC823 and SGC7901 cells, and negative control siRNA (Ne-Si) or two of HoxD10 siRNA (H-Si1 and H-Si2) in HGC27 cells. GAPDH was used as internal control. Densitometry values are expressed as fold change compared with pCDNA3.1 vector or negative control siRNA values normalized to 1. ( E ) BGC823 and SGC7901 were co-transfected with pcDNA3.1 empty vector or pcDNA3.1-HoxD10, pGL3-basic vector or pGL3-(pIGFBP3)-basic and pRL-TK vector. Relative firefly activity was expressed normalized to renilla activity in pRL-TK vector. All experiments were performed in triplicate. ** indicates of p

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, Negative Control, Activity Assay

28) Product Images from "Sp1-induced upregulation of the long noncoding RNA TINCR inhibits cell migration and invasion by regulating miR-107/miR-1286 in lung adenocarcinoma"

Article Title: Sp1-induced upregulation of the long noncoding RNA TINCR inhibits cell migration and invasion by regulating miR-107/miR-1286 in lung adenocarcinoma

Journal: American Journal of Translational Research

doi:

TINCR constrains the expression of miR-107 and miR-1286, and is a target of them in LUAD cells. A, B. Relative expressions of miR-107 and miR-1286 were analyzed by qRT-PCR analysis in A549 and NCI-H292 cells transfected with NC, si-TINCR, pcDNA3.1 and pcDNA-TINCR, respectively. C. Predicted binding sites between miR-107, D. miR-1286 and WT /MUT TINCR using the online software programs TargentScan (http://www.targetscan.org/mamm_31/) and starBase (http://starbase.sysu.edu.cn/); E, F. luciferase activity of the indicated group in A549 and NCI-H292 cells transfected with pRL-TK vector + miR-NC/miR-107/miR-1286 + TINCR WT, or pRL-TK vector + miR-NC/miR-107/miR-1286 + TINCR MUT. Data shown are mean ± standard deviation. Statistically significant differences are indicated as *, P
Figure Legend Snippet: TINCR constrains the expression of miR-107 and miR-1286, and is a target of them in LUAD cells. A, B. Relative expressions of miR-107 and miR-1286 were analyzed by qRT-PCR analysis in A549 and NCI-H292 cells transfected with NC, si-TINCR, pcDNA3.1 and pcDNA-TINCR, respectively. C. Predicted binding sites between miR-107, D. miR-1286 and WT /MUT TINCR using the online software programs TargentScan (http://www.targetscan.org/mamm_31/) and starBase (http://starbase.sysu.edu.cn/); E, F. luciferase activity of the indicated group in A549 and NCI-H292 cells transfected with pRL-TK vector + miR-NC/miR-107/miR-1286 + TINCR WT, or pRL-TK vector + miR-NC/miR-107/miR-1286 + TINCR MUT. Data shown are mean ± standard deviation. Statistically significant differences are indicated as *, P

Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Binding Assay, Software, Luciferase, Activity Assay, Plasmid Preparation, Standard Deviation

TINCR constrains the expression of miR-107 and miR-1286, and is a target of them in LUAD cells. A, B. Relative expressions of miR-107 and miR-1286 were analyzed by qRT-PCR analysis in A549 and NCI-H292 cells transfected with NC, si-TINCR, pcDNA3.1 and pcDNA-TINCR, respectively. C. Predicted binding sites between miR-107, D. miR-1286 and WT /MUT TINCR using the online software programs TargentScan (http://www.targetscan.org/mamm_31/) and starBase (http://starbase.sysu.edu.cn/); E, F. luciferase activity of the indicated group in A549 and NCI-H292 cells transfected with pRL-TK vector + miR-NC/miR-107/miR-1286 + TINCR WT, or pRL-TK vector + miR-NC/miR-107/miR-1286 + TINCR MUT. Data shown are mean ± standard deviation. Statistically significant differences are indicated as *, P
Figure Legend Snippet: TINCR constrains the expression of miR-107 and miR-1286, and is a target of them in LUAD cells. A, B. Relative expressions of miR-107 and miR-1286 were analyzed by qRT-PCR analysis in A549 and NCI-H292 cells transfected with NC, si-TINCR, pcDNA3.1 and pcDNA-TINCR, respectively. C. Predicted binding sites between miR-107, D. miR-1286 and WT /MUT TINCR using the online software programs TargentScan (http://www.targetscan.org/mamm_31/) and starBase (http://starbase.sysu.edu.cn/); E, F. luciferase activity of the indicated group in A549 and NCI-H292 cells transfected with pRL-TK vector + miR-NC/miR-107/miR-1286 + TINCR WT, or pRL-TK vector + miR-NC/miR-107/miR-1286 + TINCR MUT. Data shown are mean ± standard deviation. Statistically significant differences are indicated as *, P

Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Binding Assay, Software, Luciferase, Activity Assay, Plasmid Preparation, Standard Deviation

29) Product Images from "Whole exome sequencing identifies a novel NRL mutation in a Chinese family with autosomal dominant retinitis pigmentosa"

Article Title: Whole exome sequencing identifies a novel NRL mutation in a Chinese family with autosomal dominant retinitis pigmentosa

Journal: Molecular Vision

doi:

The p.P49L mutation in NRL increased its ability to transactivate Rho expression. WT or p.P49L mutant NRL expression constructs were co-transfected into HEK293 cells with pGL3-Rho-promoter and pRL-TK plasmids. The empty p3X-FLAG-CMV-7.1 expression vector was used as a negative control. The luciferase activities of whole cell lysates were measured. Data are the average of three replicate experiments and error bars show +/−1.00 SD. The asterisk indicates a significant difference.
Figure Legend Snippet: The p.P49L mutation in NRL increased its ability to transactivate Rho expression. WT or p.P49L mutant NRL expression constructs were co-transfected into HEK293 cells with pGL3-Rho-promoter and pRL-TK plasmids. The empty p3X-FLAG-CMV-7.1 expression vector was used as a negative control. The luciferase activities of whole cell lysates were measured. Data are the average of three replicate experiments and error bars show +/−1.00 SD. The asterisk indicates a significant difference.

Techniques Used: Mutagenesis, Expressing, Construct, Transfection, Plasmid Preparation, Negative Control, Luciferase

30) Product Images from "The lncRNA MALAT1 acts as a competing endogenous RNA to regulate KRAS expression by sponging miR-217 in pancreatic ductal adenocarcinoma"

Article Title: The lncRNA MALAT1 acts as a competing endogenous RNA to regulate KRAS expression by sponging miR-217 in pancreatic ductal adenocarcinoma

Journal: Scientific Reports

doi: 10.1038/s41598-017-05274-4

MALAT1 regulates KRAS expression in pancreatic cancer cells by interacting with miR-217. ( A ) KRAS mRNA expression after MALAT1 knockdown in pancreatic cancer cells. ( B ) KRAS and RAS protein expression after MALAT1 knockdown in pancreatic cancer cells. β-actin was used as a loading control. siCT: negative control siRNA, siM1: siMALAT1-1, siM2: siMALAT1-2. ( C ) KRAS protein expression in pancreatic cancer cells after the cells were transfected with negative-control siRNA (siCT) or siMALAT1 with or without an miR-217 mimic or miR-217 inhibitor ( D ). The full-length gels and blots for KRAS protein are included in Fig. S5 . ( E) Luciferase reporter gene expression after pRL-TK-KRAS-WT vectors or pRL-TK-KRAS-MUT vectors were transfected into pancreatic cancer cells along with either siMALAT1 or negative-control siRNA (siCT). ( F ) MALAT1 downregulation after pancreatic cancer cells were transfected with siRNAs. ( G ) XIST and GAPDH RNA levels in the nuclear and cytoplasmic fractions, as determined by qPCR. ( H ) miR-217 nucleus/cytoplasm ratios in pancreatic cancer cells after the cells were transfected with siCT or siMALAT1.
Figure Legend Snippet: MALAT1 regulates KRAS expression in pancreatic cancer cells by interacting with miR-217. ( A ) KRAS mRNA expression after MALAT1 knockdown in pancreatic cancer cells. ( B ) KRAS and RAS protein expression after MALAT1 knockdown in pancreatic cancer cells. β-actin was used as a loading control. siCT: negative control siRNA, siM1: siMALAT1-1, siM2: siMALAT1-2. ( C ) KRAS protein expression in pancreatic cancer cells after the cells were transfected with negative-control siRNA (siCT) or siMALAT1 with or without an miR-217 mimic or miR-217 inhibitor ( D ). The full-length gels and blots for KRAS protein are included in Fig. S5 . ( E) Luciferase reporter gene expression after pRL-TK-KRAS-WT vectors or pRL-TK-KRAS-MUT vectors were transfected into pancreatic cancer cells along with either siMALAT1 or negative-control siRNA (siCT). ( F ) MALAT1 downregulation after pancreatic cancer cells were transfected with siRNAs. ( G ) XIST and GAPDH RNA levels in the nuclear and cytoplasmic fractions, as determined by qPCR. ( H ) miR-217 nucleus/cytoplasm ratios in pancreatic cancer cells after the cells were transfected with siCT or siMALAT1.

Techniques Used: Expressing, Negative Control, Transfection, Luciferase, Real-time Polymerase Chain Reaction

31) Product Images from "New Compound ChlA-F Induces Autophagy-dependent Anti-cancer Effect via Upregulating Sestrin-2 in Human Bladder Cancer"

Article Title: New Compound ChlA-F Induces Autophagy-dependent Anti-cancer Effect via Upregulating Sestrin-2 in Human Bladder Cancer

Journal: Cancer letters

doi: 10.1016/j.canlet.2018.08.013

ChlA-F treatment increased both SESN2 transcription and SESN2 mRNA stability. ( A ) Total RNAs were isolated from T24T cells treated with or without 8 µM ChlA-F for the indicated times. RT-PCR was performed to determine SESN2 mRNA levels. The gapdh mRNA levels were used as loading control. ( B ) T24T stably co-transfected with SESN2 promoter-driven luciferase reporter and pRL -TK was treated with 8µM ChlA-F for the indicated times to evaluate SESN2 promoter transcriptional activity. The induction fold was normalized to internal control pRL-TK . The results were presented as SESN2 promoter activity relative to vehicle control (relative SESN2 promoter activity). The bars indicate a Mean ± SD from three independent experiments. The symbol (*) indicates a significant increase from the vehicle control ( p
Figure Legend Snippet: ChlA-F treatment increased both SESN2 transcription and SESN2 mRNA stability. ( A ) Total RNAs were isolated from T24T cells treated with or without 8 µM ChlA-F for the indicated times. RT-PCR was performed to determine SESN2 mRNA levels. The gapdh mRNA levels were used as loading control. ( B ) T24T stably co-transfected with SESN2 promoter-driven luciferase reporter and pRL -TK was treated with 8µM ChlA-F for the indicated times to evaluate SESN2 promoter transcriptional activity. The induction fold was normalized to internal control pRL-TK . The results were presented as SESN2 promoter activity relative to vehicle control (relative SESN2 promoter activity). The bars indicate a Mean ± SD from three independent experiments. The symbol (*) indicates a significant increase from the vehicle control ( p

Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transfection, Luciferase, Activity Assay

32) Product Images from "Transcriptional activation of the anchoring protein SAP97 by heat shock factor (HSF)-1 stabilizes Kv1.5 channels in HL-1 cells"

Article Title: Transcriptional activation of the anchoring protein SAP97 by heat shock factor (HSF)-1 stabilizes Kv1.5 channels in HL-1 cells

Journal: British Journal of Pharmacology

doi: 10.1111/j.1476-5381.2011.01204.x

HS regulation of the activity of SAP97 promoter with HSEs. Effects of HS on luciferase activity in HL-1 cells transfected with reporter gene. Cells were co-transfected with SAP97 gene ∼1 Kb promoter-pGL3Basic luciferase reporter fusion plasmid containing (pGL3/SAP97) or lacking HSEs (pGL3/ΔHSEs) and pRL-TK vector as internal control; luciferase activities were measured after stimulation with HS (at 42°C for 30 min). The mean ratio (±SEM) of the firefly luciferase activity normalized to Renilla luciferase per well ( n = 20 for pGL3 vector, pGL3/SAP97 and pGL3/ΔHSEs groups) is shown. Statistical significance of the differences among each group was tested by Student's t -test: * P
Figure Legend Snippet: HS regulation of the activity of SAP97 promoter with HSEs. Effects of HS on luciferase activity in HL-1 cells transfected with reporter gene. Cells were co-transfected with SAP97 gene ∼1 Kb promoter-pGL3Basic luciferase reporter fusion plasmid containing (pGL3/SAP97) or lacking HSEs (pGL3/ΔHSEs) and pRL-TK vector as internal control; luciferase activities were measured after stimulation with HS (at 42°C for 30 min). The mean ratio (±SEM) of the firefly luciferase activity normalized to Renilla luciferase per well ( n = 20 for pGL3 vector, pGL3/SAP97 and pGL3/ΔHSEs groups) is shown. Statistical significance of the differences among each group was tested by Student's t -test: * P

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

33) Product Images from "Melatonin reverses nasopharyngeal carcinoma cisplatin chemoresistance by inhibiting the Wnt/β-catenin signaling pathway"

Article Title: Melatonin reverses nasopharyngeal carcinoma cisplatin chemoresistance by inhibiting the Wnt/β-catenin signaling pathway

Journal: Aging (Albany NY)

doi: 10.18632/aging.102968

Melatonin reverses DDP chemoresistance by inhibiting β-catenin nuclear translocation. ( A , B ) Protein expression of β-catenin, DKK1, c-Myc and CylincD1 in the CNE2 and CNE2/DDP ( A ), 5-8F and 5-8F/DDP cell lines ( B ) treated with melatonin (2 mM) for 48 hr, as determined by western blot. ( C ) mRNA expression of Wnt/β-catenin downstream genes (AXIN2, SOX9, CD44 and CCND2) in CNE2 and CNE2/DDP, 5-8F and 5-8F/DDP cell lines treated with melatonin (2 mM) for 48 hr, as determined by qPCR. ( D , E ) Representative images of immunofluorescent staining for β-catenin in CNE2 and CNE2/DDP ( D ), 5-8F and 5-8F/DDP ( E ) treated with or without melatonin (2 mM) for 48 hr. ( F ) Relative luciferase activity of NPC cells transfected with the TOP/FOPFlash vector and pRL-TK vector. Data presented are the mean ± SD; ** P
Figure Legend Snippet: Melatonin reverses DDP chemoresistance by inhibiting β-catenin nuclear translocation. ( A , B ) Protein expression of β-catenin, DKK1, c-Myc and CylincD1 in the CNE2 and CNE2/DDP ( A ), 5-8F and 5-8F/DDP cell lines ( B ) treated with melatonin (2 mM) for 48 hr, as determined by western blot. ( C ) mRNA expression of Wnt/β-catenin downstream genes (AXIN2, SOX9, CD44 and CCND2) in CNE2 and CNE2/DDP, 5-8F and 5-8F/DDP cell lines treated with melatonin (2 mM) for 48 hr, as determined by qPCR. ( D , E ) Representative images of immunofluorescent staining for β-catenin in CNE2 and CNE2/DDP ( D ), 5-8F and 5-8F/DDP ( E ) treated with or without melatonin (2 mM) for 48 hr. ( F ) Relative luciferase activity of NPC cells transfected with the TOP/FOPFlash vector and pRL-TK vector. Data presented are the mean ± SD; ** P

Techniques Used: Translocation Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Staining, Luciferase, Activity Assay, Transfection, Plasmid Preparation

34) Product Images from "PGRP negatively regulates NOD-mediated cytokine production in rainbow trout liver cells"

Article Title: PGRP negatively regulates NOD-mediated cytokine production in rainbow trout liver cells

Journal: Scientific Reports

doi: 10.1038/srep39344

The effect of secreted OmPGRP-L1 on the NOD-induced NF-κB pathway in iE-DAP- and MDP-stimulated RTH-149 cells. ( a ) The secretion of OmPGRP-L1-FLAG recombinant protein in the conditioned media was confirmed by western blotting with a mouse monoclonal anti-FLAG antibody. ( b ) RTH-149 cells were cultured in 6-well plates, and the cell culture medium was replaced with the conditioned media containing the secreted OmPGRP-L1-FLAG recombinant protein (OmPGRP-L1 CM) or empty vector CM. Next, cells were stimulated with 10 μg/ml iE-DAP and 100 μg/ml MDP for 24 h, and the expression of pro-inflammatory cytokines was analysed by qRT-PCR. ( c ) RTH-149 cells were co-transfected with pNF-κB-Luc and pRL-TK vectors. At 48 h after transfection, the cell culture medium was replaced with conditioned media. Next, cells were stimulated with 10 μg/ml iE-DAP and 100 μg/ml MDP for 24 h, and NF-κB activity was measured as described in the Methods. ( d ) RTH-149 cells were stimulated with iE-DAP (10 μg/ml) and MDP (100 μg/ml) for the indicated times. Next, the phospho- and total forms of IκBα and TAK1 were assessed by western blotting. ( e , f ) RTH-149 cells were transfected with pcDNA3.1-OmPGRP-L1-FLAG, empty pcDNA3.1 vector, OmPGRP-L1 siRNA or scrambled-siRNA. At 48 h after transfection, cells (except control cells) were stimulated with iE-DAP (10 μg/ml) and MDP (100 μg/ml) for 6 h. Next, the phospho- and total forms of IκBα and TAK1 were assessed by western blotting. The data in ( b ) and ( c ) are shown as the mean ± SEM of three independent experiments performed in triplicate. *P
Figure Legend Snippet: The effect of secreted OmPGRP-L1 on the NOD-induced NF-κB pathway in iE-DAP- and MDP-stimulated RTH-149 cells. ( a ) The secretion of OmPGRP-L1-FLAG recombinant protein in the conditioned media was confirmed by western blotting with a mouse monoclonal anti-FLAG antibody. ( b ) RTH-149 cells were cultured in 6-well plates, and the cell culture medium was replaced with the conditioned media containing the secreted OmPGRP-L1-FLAG recombinant protein (OmPGRP-L1 CM) or empty vector CM. Next, cells were stimulated with 10 μg/ml iE-DAP and 100 μg/ml MDP for 24 h, and the expression of pro-inflammatory cytokines was analysed by qRT-PCR. ( c ) RTH-149 cells were co-transfected with pNF-κB-Luc and pRL-TK vectors. At 48 h after transfection, the cell culture medium was replaced with conditioned media. Next, cells were stimulated with 10 μg/ml iE-DAP and 100 μg/ml MDP for 24 h, and NF-κB activity was measured as described in the Methods. ( d ) RTH-149 cells were stimulated with iE-DAP (10 μg/ml) and MDP (100 μg/ml) for the indicated times. Next, the phospho- and total forms of IκBα and TAK1 were assessed by western blotting. ( e , f ) RTH-149 cells were transfected with pcDNA3.1-OmPGRP-L1-FLAG, empty pcDNA3.1 vector, OmPGRP-L1 siRNA or scrambled-siRNA. At 48 h after transfection, cells (except control cells) were stimulated with iE-DAP (10 μg/ml) and MDP (100 μg/ml) for 6 h. Next, the phospho- and total forms of IκBα and TAK1 were assessed by western blotting. The data in ( b ) and ( c ) are shown as the mean ± SEM of three independent experiments performed in triplicate. *P

Techniques Used: Recombinant, Western Blot, Cell Culture, Plasmid Preparation, Expressing, Quantitative RT-PCR, Transfection, Activity Assay

35) Product Images from "Association between functional polymorphisms in IL‐33/ST2 pathway and risk of osteosarcoma. Association between functional polymorphisms in IL‐33/ST2 pathway and risk of osteosarcoma"

Article Title: Association between functional polymorphisms in IL‐33/ST2 pathway and risk of osteosarcoma. Association between functional polymorphisms in IL‐33/ST2 pathway and risk of osteosarcoma

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13653

Dual‐luciferase reporter gene analysis. The human osteosarcoma ( OS ) cell lines ( MG ‐63 and Saos‐2) and the human embryonic kidney cell line 293 ( HEK 293) were transfected simultaneously with rs7025417T or rs7025417C plasmid and pRL ‐ TK vector. The luciferase activity was measured using the dual luciferase reporter assay. Data were expressed by mean ± standard deviation (** P
Figure Legend Snippet: Dual‐luciferase reporter gene analysis. The human osteosarcoma ( OS ) cell lines ( MG ‐63 and Saos‐2) and the human embryonic kidney cell line 293 ( HEK 293) were transfected simultaneously with rs7025417T or rs7025417C plasmid and pRL ‐ TK vector. The luciferase activity was measured using the dual luciferase reporter assay. Data were expressed by mean ± standard deviation (** P

Techniques Used: Luciferase, Transfection, Plasmid Preparation, Activity Assay, Reporter Assay, Standard Deviation

36) Product Images from "A Functional Insertion/Deletion Polymorphism in the Proximal Promoter of CD3G Is Associated with Susceptibility for Hepatocellular Carcinoma in Chinese Population"

Article Title: A Functional Insertion/Deletion Polymorphism in the Proximal Promoter of CD3G Is Associated with Susceptibility for Hepatocellular Carcinoma in Chinese Population

Journal: DNA and Cell Biology

doi: 10.1089/dna.2012.1706

Luciferase expression of two constructs in HepG2 and sk-Hep-1 cells co-transfected with pRL-TK to standardize transfection efficiency. * p
Figure Legend Snippet: Luciferase expression of two constructs in HepG2 and sk-Hep-1 cells co-transfected with pRL-TK to standardize transfection efficiency. * p

Techniques Used: Luciferase, Expressing, Construct, Transfection

37) Product Images from "Induction of SOCS3 by liver X receptor suppresses the proliferation of hepatocellular carcinoma cells"

Article Title: Induction of SOCS3 by liver X receptor suppresses the proliferation of hepatocellular carcinoma cells

Journal: Oncotarget

doi: 10.18632/oncotarget.19321

Activation of LXR enhances the stability of SOCS3 mRNA (A) The potential LXREs in human SOCS3 gene promoter region were predicted using bioinformatic analysis ( http://www.nubiscan.unibas.ch/ ). (B, C) After transiently transfected with pRL-TK and pGL3-SOCS3 (or the control pGL3-basic) for 12 h, HepG2 cells were treated with 2 μM GW4064 or 5 μM TO901317 (or DMSO) for 18 h. Then the luciferase activity of SOCS3 gene promoter region was detected by dual luciferase reporter assay system. Data represent the mean ± SD of triplicate experiments. (D) After treated with DMSO or TO90137 (5 μM) for 12 h, HepG2 cells were incubated with actinomycin D (Act D, 10 μg/ml) for the indicated times. Then the mRNA level of SOCS3 was examined by qPCR. (E) HepG2 cells were treated with TO901317 (5 μM) for 12 h, followed by the incubation with 50 μg/ml cycloheximide (CHX) for the indicated times. Then the level of SOCS3 protein was assayed by Western blot. (F) The quantitative analysis of the Western blot results in (E) using quantity-one software. The relative value of SOCS3 protein level was normalized by the control tubulin. * P
Figure Legend Snippet: Activation of LXR enhances the stability of SOCS3 mRNA (A) The potential LXREs in human SOCS3 gene promoter region were predicted using bioinformatic analysis ( http://www.nubiscan.unibas.ch/ ). (B, C) After transiently transfected with pRL-TK and pGL3-SOCS3 (or the control pGL3-basic) for 12 h, HepG2 cells were treated with 2 μM GW4064 or 5 μM TO901317 (or DMSO) for 18 h. Then the luciferase activity of SOCS3 gene promoter region was detected by dual luciferase reporter assay system. Data represent the mean ± SD of triplicate experiments. (D) After treated with DMSO or TO90137 (5 μM) for 12 h, HepG2 cells were incubated with actinomycin D (Act D, 10 μg/ml) for the indicated times. Then the mRNA level of SOCS3 was examined by qPCR. (E) HepG2 cells were treated with TO901317 (5 μM) for 12 h, followed by the incubation with 50 μg/ml cycloheximide (CHX) for the indicated times. Then the level of SOCS3 protein was assayed by Western blot. (F) The quantitative analysis of the Western blot results in (E) using quantity-one software. The relative value of SOCS3 protein level was normalized by the control tubulin. * P

Techniques Used: Activation Assay, Transfection, Luciferase, Activity Assay, Reporter Assay, Incubation, Activated Clotting Time Assay, Real-time Polymerase Chain Reaction, Western Blot, Software

38) Product Images from "A plasmid containing CpG ODN as vaccine adjuvant against grass carp reovirus in grass carp Ctenopharyngodon idella"

Article Title: A plasmid containing CpG ODN as vaccine adjuvant against grass carp reovirus in grass carp Ctenopharyngodon idella

Journal: Oncotarget

doi: 10.18632/oncotarget.21245

Immune responses of CiIFN1/3 to plasmids containing CpG ODNs Left, the mRNA expressions of CiIFN1 (A) and CiIFN3 (B) were measured at 24 and 48 h post-infection. CIK cells were stimulated with PBS (control) or plasmids containing CpG ODNs and infected with GCRV. Other captions were the same as Figure 3 . Right, CIK cells were co-transfected with 800 ng of pRL-TK and IFN1pro-luc (A) or IFN3pro-luc (B) in 24-well plates. At 16 h post-transfection, the cells were stimulated with PBS (control) or plasmids and infected with GCRV for 16 h or uninfected. Dual luciferase reporter assays were conducted at 12 h after GCRV infection. Treatment durations in this assay was far shorter than those in qRT-PCR which caused different results. Error bars indicate standard deviation (n = 4). Asterisks indicate significant difference from control (*, P ≤ 0.05).
Figure Legend Snippet: Immune responses of CiIFN1/3 to plasmids containing CpG ODNs Left, the mRNA expressions of CiIFN1 (A) and CiIFN3 (B) were measured at 24 and 48 h post-infection. CIK cells were stimulated with PBS (control) or plasmids containing CpG ODNs and infected with GCRV. Other captions were the same as Figure 3 . Right, CIK cells were co-transfected with 800 ng of pRL-TK and IFN1pro-luc (A) or IFN3pro-luc (B) in 24-well plates. At 16 h post-transfection, the cells were stimulated with PBS (control) or plasmids and infected with GCRV for 16 h or uninfected. Dual luciferase reporter assays were conducted at 12 h after GCRV infection. Treatment durations in this assay was far shorter than those in qRT-PCR which caused different results. Error bars indicate standard deviation (n = 4). Asterisks indicate significant difference from control (*, P ≤ 0.05).

Techniques Used: Infection, Transfection, Luciferase, Quantitative RT-PCR, Standard Deviation

39) Product Images from "NNK, a Tobacco-Specific Carcinogen, Inhibits the Expression of Lysyl Oxidase, a Tumor Suppressor"

Article Title: NNK, a Tobacco-Specific Carcinogen, Inhibits the Expression of Lysyl Oxidase, a Tumor Suppressor

Journal: International Journal of Environmental Research and Public Health

doi: 10.3390/ijerph120100064

Inhibition of LOX promoter activities in NNK-treated cells. ( A ) Schematic representation of LOX promoter-reporter chimera with the Inr-DPE core promoter. ( B ) NNK inhibition of LOX promoter activities in transfected cells. RFL6 cells were transiently co-transfected with the Prom-804 construct and the pRL-TK vector, an internal control, then treated with NNK at indicated concentrations for 48 h. Luciferase activities in cell lysates were measured by luminometry. Firefly luciferase activities elicited by the LOX promoter were normalized to Renilla luciferase activities derived from the pRL-TK vector and expressed as relative luciferase activities as instructed by manufacturer (Promega). Data shown are the mean ± SD ( n = 3). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared with the control (100%).
Figure Legend Snippet: Inhibition of LOX promoter activities in NNK-treated cells. ( A ) Schematic representation of LOX promoter-reporter chimera with the Inr-DPE core promoter. ( B ) NNK inhibition of LOX promoter activities in transfected cells. RFL6 cells were transiently co-transfected with the Prom-804 construct and the pRL-TK vector, an internal control, then treated with NNK at indicated concentrations for 48 h. Luciferase activities in cell lysates were measured by luminometry. Firefly luciferase activities elicited by the LOX promoter were normalized to Renilla luciferase activities derived from the pRL-TK vector and expressed as relative luciferase activities as instructed by manufacturer (Promega). Data shown are the mean ± SD ( n = 3). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared with the control (100%).

Techniques Used: Inhibition, Transfection, Construct, Plasmid Preparation, Luciferase, Derivative Assay

40) Product Images from "Dominant ER Stress–Inducing WFS1 Mutations Underlie a Genetic Syndrome of Neonatal/Infancy-Onset Diabetes, Congenital Sensorineural Deafness, and Congenital Cataracts"

Article Title: Dominant ER Stress–Inducing WFS1 Mutations Underlie a Genetic Syndrome of Neonatal/Infancy-Onset Diabetes, Congenital Sensorineural Deafness, and Congenital Cataracts

Journal: Diabetes

doi: 10.2337/db16-1296

Luciferase reporter assays in HeLa cells transfected with the ERSE reporter together with control (pcDNA) (EV), wild-type WFS1 (WT), mutant c.2171C > T p.Pro724Leu (P724L), mutant c.937C > T p.His313Tyr (p.H313Y), mutant c.2489A > C p.Glu830Ala (p.E830A), or mutant c.2425G > A p.Glu809Lys (p.E809K) expression plasmid. Cells were untreated (UT) or treated with TG (100 nmol/L) for 8 h. Relative intensity of luciferase (Promega Dual-Luciferase Reporter Assay System) was then measured ( n = 3; dashed lines represent mean). Transfections were normalized with the pRL-TK vector (Promega) as an internal control. Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett test: * P
Figure Legend Snippet: Luciferase reporter assays in HeLa cells transfected with the ERSE reporter together with control (pcDNA) (EV), wild-type WFS1 (WT), mutant c.2171C > T p.Pro724Leu (P724L), mutant c.937C > T p.His313Tyr (p.H313Y), mutant c.2489A > C p.Glu830Ala (p.E830A), or mutant c.2425G > A p.Glu809Lys (p.E809K) expression plasmid. Cells were untreated (UT) or treated with TG (100 nmol/L) for 8 h. Relative intensity of luciferase (Promega Dual-Luciferase Reporter Assay System) was then measured ( n = 3; dashed lines represent mean). Transfections were normalized with the pRL-TK vector (Promega) as an internal control. Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett test: * P

Techniques Used: Luciferase, Transfection, Mutagenesis, Expressing, Plasmid Preparation, Reporter Assay

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Amplification:

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Agarose Gel Electrophoresis:

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Article Snippet: .. In the case of mixed infections (presence of double peaks in the analyzed pherograms from the sequencing of the original PCR amplicon), purified PCR products were ligated into pGEM-T easy cloning vector following the manufacturer's instructions (pGEM-T clone kit; Promega, Madison, WI) and transformed into Escherichia coli DH5α competent cells by heat shock. .. Positive colonies were selected by blue/white screening followed by colony PCR using M13F/R primers under the following conditions: 5 min at 95°C and 35 cycles of 60 s at 95°C, 60 s at 51°C, and 1 min and 20 s at 72°C and a final extension of 5 min at 72°C.

Article Title: Motifs within the CA-repeat-rich region of Surfactant Protein B (SFTPB) intron 4 differentially affect mRNA splicing
Article Snippet: .. To improve the specificity of amplification in order to generate different length variants, the following touchdown PCR program ( ) was used: 95°C for 2 min, 5 cycles of 95°C for 30 s, 70°C for 30 s and 68°C for 30 s, then 5 cycles of 95°C for 30 s, 68°C for 30 s and 68°C for 30 s, and then 5 cycles of 95°C for 30 s, 66°C for 30 s and 68°C for 30 s, followed by 25 cycles of 94°C for 30 s, 64°C for 30 s and 68°C for 30 s and a final extension step at 68°C for 5 min. All PCR fragments, ranging from 96 to 437 were separated on agarose gel, purified and cloned into a pGEM-T vector (Promega, WI, USA). .. The recombinant plasmid DNAs were digested, purified and sub-cloned into the EcoR I and Not I sites of pcDNA 3.1 minigene Del-m ( , inset).

Electrophoresis:

Article Title: Cloning and molecular characterization of Δ12-fatty acid desaturase gene from Mortierella isabellina
Article Snippet: .. PCR product about 1.2 kb long was gel-purified by electrophoresis and cloned into pGEM-T vector. .. Positive clones were selected on LB plate by color reaction.

Polymerase Chain Reaction:

Article Title: Engineering Saccharomyces cerevisiae for improvement in ethanol tolerance by accumulation of trehalose
Article Snippet: .. The 5′ UP nth1 -kanMX-3′ DOWN nth1 , obtained from the third round of PCR, was ligated with pGEMT vector (Promega Corporation, Madison, WI, USA) and transformed into CaCl2 -treated E. coli TOP10F’ according to Hanahan and Meselson. .. The transformed host was screened by blue/white selection.

Article Title: Octamer binding protein 2 (Oct2) regulates PD-L2 gene expression in B-1 cells through lineage-specific activity of a unique, intronic promoter
Article Snippet: .. After checking the band pattern, PCR products were TA-cloned into pGEM-T vector (Promega, Madison, WI, USA). ..

Article Title: Cloning and molecular characterization of Δ12-fatty acid desaturase gene from Mortierella isabellina
Article Snippet: .. PCR product about 1.2 kb long was gel-purified by electrophoresis and cloned into pGEM-T vector. .. Positive clones were selected on LB plate by color reaction.

Article Title: Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale
Article Snippet: .. In the case of mixed infections (presence of double peaks in the analyzed pherograms from the sequencing of the original PCR amplicon), purified PCR products were ligated into pGEM-T easy cloning vector following the manufacturer's instructions (pGEM-T clone kit; Promega, Madison, WI) and transformed into Escherichia coli DH5α competent cells by heat shock. .. Positive colonies were selected by blue/white screening followed by colony PCR using M13F/R primers under the following conditions: 5 min at 95°C and 35 cycles of 60 s at 95°C, 60 s at 51°C, and 1 min and 20 s at 72°C and a final extension of 5 min at 72°C.

Touchdown PCR:

Article Title: Motifs within the CA-repeat-rich region of Surfactant Protein B (SFTPB) intron 4 differentially affect mRNA splicing
Article Snippet: .. To improve the specificity of amplification in order to generate different length variants, the following touchdown PCR program ( ) was used: 95°C for 2 min, 5 cycles of 95°C for 30 s, 70°C for 30 s and 68°C for 30 s, then 5 cycles of 95°C for 30 s, 68°C for 30 s and 68°C for 30 s, and then 5 cycles of 95°C for 30 s, 66°C for 30 s and 68°C for 30 s, followed by 25 cycles of 94°C for 30 s, 64°C for 30 s and 68°C for 30 s and a final extension step at 68°C for 5 min. All PCR fragments, ranging from 96 to 437 were separated on agarose gel, purified and cloned into a pGEM-T vector (Promega, WI, USA). .. The recombinant plasmid DNAs were digested, purified and sub-cloned into the EcoR I and Not I sites of pcDNA 3.1 minigene Del-m ( , inset).

Sequencing:

Article Title: Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale
Article Snippet: .. In the case of mixed infections (presence of double peaks in the analyzed pherograms from the sequencing of the original PCR amplicon), purified PCR products were ligated into pGEM-T easy cloning vector following the manufacturer's instructions (pGEM-T clone kit; Promega, Madison, WI) and transformed into Escherichia coli DH5α competent cells by heat shock. .. Positive colonies were selected by blue/white screening followed by colony PCR using M13F/R primers under the following conditions: 5 min at 95°C and 35 cycles of 60 s at 95°C, 60 s at 51°C, and 1 min and 20 s at 72°C and a final extension of 5 min at 72°C.

Transformation Assay:

Article Title: Engineering Saccharomyces cerevisiae for improvement in ethanol tolerance by accumulation of trehalose
Article Snippet: .. The 5′ UP nth1 -kanMX-3′ DOWN nth1 , obtained from the third round of PCR, was ligated with pGEMT vector (Promega Corporation, Madison, WI, USA) and transformed into CaCl2 -treated E. coli TOP10F’ according to Hanahan and Meselson. .. The transformed host was screened by blue/white selection.

Article Title: Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale
Article Snippet: .. In the case of mixed infections (presence of double peaks in the analyzed pherograms from the sequencing of the original PCR amplicon), purified PCR products were ligated into pGEM-T easy cloning vector following the manufacturer's instructions (pGEM-T clone kit; Promega, Madison, WI) and transformed into Escherichia coli DH5α competent cells by heat shock. .. Positive colonies were selected by blue/white screening followed by colony PCR using M13F/R primers under the following conditions: 5 min at 95°C and 35 cycles of 60 s at 95°C, 60 s at 51°C, and 1 min and 20 s at 72°C and a final extension of 5 min at 72°C.

Plasmid Preparation:

Article Title: Chromosome-Based Genetic Complementation System for Xylella fastidiosa ▿
Article Snippet: .. The resulting 1.6-kb fragment was then cloned into the pGEM-T vector (Promega), creating plasmid pAX1. .. PCR was also used to generate DNA fragments carrying the multiple cloning sites and the four antibiotic resistance cassettes that were inserted into pAX1.

Article Title: Engineering Saccharomyces cerevisiae for improvement in ethanol tolerance by accumulation of trehalose
Article Snippet: .. The 5′ UP nth1 -kanMX-3′ DOWN nth1 , obtained from the third round of PCR, was ligated with pGEMT vector (Promega Corporation, Madison, WI, USA) and transformed into CaCl2 -treated E. coli TOP10F’ according to Hanahan and Meselson. .. The transformed host was screened by blue/white selection.

Article Title: Octamer binding protein 2 (Oct2) regulates PD-L2 gene expression in B-1 cells through lineage-specific activity of a unique, intronic promoter
Article Snippet: .. After checking the band pattern, PCR products were TA-cloned into pGEM-T vector (Promega, Madison, WI, USA). ..

Article Title: Cloning and molecular characterization of Δ12-fatty acid desaturase gene from Mortierella isabellina
Article Snippet: .. PCR product about 1.2 kb long was gel-purified by electrophoresis and cloned into pGEM-T vector. .. Positive clones were selected on LB plate by color reaction.

Article Title: Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale
Article Snippet: .. In the case of mixed infections (presence of double peaks in the analyzed pherograms from the sequencing of the original PCR amplicon), purified PCR products were ligated into pGEM-T easy cloning vector following the manufacturer's instructions (pGEM-T clone kit; Promega, Madison, WI) and transformed into Escherichia coli DH5α competent cells by heat shock. .. Positive colonies were selected by blue/white screening followed by colony PCR using M13F/R primers under the following conditions: 5 min at 95°C and 35 cycles of 60 s at 95°C, 60 s at 51°C, and 1 min and 20 s at 72°C and a final extension of 5 min at 72°C.

Article Title: Two RNA editing sites with cis-acting elements of moderate sequence identity are recognized by an identical site-recognition protein in tobacco chloroplasts
Article Snippet: .. The amplified fragments were cloned into a pGEM-T vector using the pGEM-T Vector System (Promega). .. From these cloned plasmids, RNA substrates for in vitro editing and UV-crosslinking were prepared as previously described ( ) with slight modifications.

Article Title: A gene encoding a protein modified by the phytohormone indoleacetic acid
Article Snippet: .. The iap1 cDNA was cloned into a pGEM-T vector and was used for coupled in vitro transcription and translation using the TnT Quick Coupled System (Promega) with [35 S]methionine. .. GeneRacer Kit (Invitrogen) was used for full-length, RNA ligase-mediated rapid amplification of 5′ ends using RNA isolated 24 days after flowering.

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  • 94
    Promega prl tk vector
    Involvement of PPARα on the cyanidin-induced elevation of TRPM6 and CNNM4 promoter activities. TRPM6 promoter ( A ) or CNNM4 promoter ( B ) luciferase vectors were co-transfected with a <t>pRL-TK</t> vector into MCE301 cells. WT and mutant indicate vectors with wild-type and mutant PPRE sequences, respectively. At 24 h after <t>transfection,</t> the cells were incubated with vehicle, 10 μM cyanidin, cyanidin plus 10 μM GW6471, or cyanidin plus 10 μM GW9662 for 6 h. The relative promoter activity was represented relative to the values in vehicle. n = 3–4. ** p
    Prl Tk Vector, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 1212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/prl tk vector/product/Promega
    Average 94 stars, based on 1212 article reviews
    Price from $9.99 to $1999.99
    prl tk vector - by Bioz Stars, 2020-08
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    93
    Promega prl tk renilla plasmid
    An SP1 site located at the MDK promoter is necessary for MDK promoter activation in glioma cell lines. ( A) SP1-knockdown stable U87 and U251 cell lines were transfected with MDK (-562/+33)-luc or pGL-3 Bacis vector and <t>pRL-TK.</t> 24 h after transfection, the levels of luciferase activity were measured and normalized to <t>Renilla</t> luciferase activity. (B) SP1 overexpression and control A172 cell lines were transfected with MDK (−562/+33)-luc or pGL-3 Bacis vector and pRL-TK. At 24 h after transfection, the levels of luciferase activity were measured. (C) Structures of MDK 5′ sequential deletion constructs. U87 cells were cotransfected with reporter plasmids and pRL-TK for 24 h. Promoter activity was determined by luciferase assay. (D) DNA isolated and purified from ChIP immunoprecipitated material was amplified by PCR with primers to amplify an MDK promoter fragment spanning the SP1 site; equal amounts of total genomic DNA were used for input. Data are representative of three separate experiments.
    Prl Tk Renilla Plasmid, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 236 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/prl tk renilla plasmid/product/Promega
    Average 93 stars, based on 236 article reviews
    Price from $9.99 to $1999.99
    prl tk renilla plasmid - by Bioz Stars, 2020-08
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    90
    Promega prl tk plasmids
    miR-4666-3p targets TGFBR1 in CRC cells. (A) Bioinformatics analysis of potential targets of miR-4666-3p; (B) TGFBR1 is predicted to be a novel target of miR-4666-3p; (C) 293T cells were <t>cotransfected</t> with empty pmirGLO Dual-Luciferase reporter plasmids or TGFBR1 3′UTR firefly luciferase reporter plasmids and <t>pRL-TK-luciferase</t> plasmids, together with the miR-329 or anti-miR-4666-3p mimic. After 48 h, firefly luciferase activity was measured and normalized to that of Renilla luciferase; (D) CRC cells were transfected with the NC, miR-4666-3p or anti-miR-4666-3p mimics, and expression of TGFBR1 was detected by Western blotting; (E) The activity of the TGF-β/Smad pathway was measured by the SBE luciferase reporter system in colon cancer cells (presented as P1 cells) with distinct treatment combination; (F) Serial sphere formation with different cells (control, miR-4666-3p, anti-miR-4666-3p stable expression and both miR-4666-3p and TGF-βR1 stable expression cells). The data are shown as one typical result from three independent experiments with similar results or as the mean ± SD of three independent experiments. * P
    Prl Tk Plasmids, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/prl tk plasmids/product/Promega
    Average 90 stars, based on 12 article reviews
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    Involvement of PPARα on the cyanidin-induced elevation of TRPM6 and CNNM4 promoter activities. TRPM6 promoter ( A ) or CNNM4 promoter ( B ) luciferase vectors were co-transfected with a pRL-TK vector into MCE301 cells. WT and mutant indicate vectors with wild-type and mutant PPRE sequences, respectively. At 24 h after transfection, the cells were incubated with vehicle, 10 μM cyanidin, cyanidin plus 10 μM GW6471, or cyanidin plus 10 μM GW9662 for 6 h. The relative promoter activity was represented relative to the values in vehicle. n = 3–4. ** p

    Journal: Nutrients

    Article Title: Cyanidin Increases the Expression of Mg2+ Transport Carriers Mediated by the Activation of PPARα in Colonic Epithelial MCE301 Cells

    doi: 10.3390/nu11030641

    Figure Lengend Snippet: Involvement of PPARα on the cyanidin-induced elevation of TRPM6 and CNNM4 promoter activities. TRPM6 promoter ( A ) or CNNM4 promoter ( B ) luciferase vectors were co-transfected with a pRL-TK vector into MCE301 cells. WT and mutant indicate vectors with wild-type and mutant PPRE sequences, respectively. At 24 h after transfection, the cells were incubated with vehicle, 10 μM cyanidin, cyanidin plus 10 μM GW6471, or cyanidin plus 10 μM GW9662 for 6 h. The relative promoter activity was represented relative to the values in vehicle. n = 3–4. ** p

    Article Snippet: A Renilla construct, pRL-TK vector (Promega), was used for normalizing transfection efficiency.

    Techniques: Luciferase, Transfection, Plasmid Preparation, Mutagenesis, Incubation, Activity Assay

    Beclin 1 is a direct target of miR-30a in U251 cells. (A) Bioinformatic prediction data indicated that beclin 1 was a direct target gene of miR-30a. (B and C) WT or MUT BECN1 3′-UTR was cloned downstream of the firefly luciferase coding region of the pmirGLO™ vector, to form pMIR-WT BECN1 and pMIR-MUT BECN1, respectively. (D) U251 cells were co-transfected with pMIR-WT BECN1 or pMIR-MUT BECN1 vector and miR-30a mimic or miR-NC, and pRL-TK plasmid for internal normalization, respectively. Luciferase reporter assay data showed that co-transfection with pMIR-WT BECN1 and miR-30a mimic significantly decreased the luciferase activity; however, co-transfection with pMIR-MUT BECN1 and miR-30a mimic caused no change in luciferase activity. Control cells were transfected with pMIR-WT BECN1 or pMIR-MUT BECN1 vector and the pRL-TK plasmid. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: MicroRNA-30a increases the chemosensitivity of U251 glioblastoma cells to temozolomide by directly targeting beclin 1 and inhibiting autophagy

    doi: 10.3892/etm.2018.6007

    Figure Lengend Snippet: Beclin 1 is a direct target of miR-30a in U251 cells. (A) Bioinformatic prediction data indicated that beclin 1 was a direct target gene of miR-30a. (B and C) WT or MUT BECN1 3′-UTR was cloned downstream of the firefly luciferase coding region of the pmirGLO™ vector, to form pMIR-WT BECN1 and pMIR-MUT BECN1, respectively. (D) U251 cells were co-transfected with pMIR-WT BECN1 or pMIR-MUT BECN1 vector and miR-30a mimic or miR-NC, and pRL-TK plasmid for internal normalization, respectively. Luciferase reporter assay data showed that co-transfection with pMIR-WT BECN1 and miR-30a mimic significantly decreased the luciferase activity; however, co-transfection with pMIR-MUT BECN1 and miR-30a mimic caused no change in luciferase activity. Control cells were transfected with pMIR-WT BECN1 or pMIR-MUT BECN1 vector and the pRL-TK plasmid. *P

    Article Snippet: After that, U251 cells were co-transfected with pMIR-WT BECN1 or pMIR-MUT BECN1 vector and miR-30a mimic or scrambled miR mimic, and the pRL-TK plasmid (Promega Corporation) for internal normalization, respectively, and cultured for 48 h. The transfected cells were then lysed using lysis buffer (Promega Corporation).

    Techniques: Clone Assay, Luciferase, Plasmid Preparation, Transfection, Reporter Assay, Cotransfection, Activity Assay

    An SP1 site located at the MDK promoter is necessary for MDK promoter activation in glioma cell lines. ( A) SP1-knockdown stable U87 and U251 cell lines were transfected with MDK (-562/+33)-luc or pGL-3 Bacis vector and pRL-TK. 24 h after transfection, the levels of luciferase activity were measured and normalized to Renilla luciferase activity. (B) SP1 overexpression and control A172 cell lines were transfected with MDK (−562/+33)-luc or pGL-3 Bacis vector and pRL-TK. At 24 h after transfection, the levels of luciferase activity were measured. (C) Structures of MDK 5′ sequential deletion constructs. U87 cells were cotransfected with reporter plasmids and pRL-TK for 24 h. Promoter activity was determined by luciferase assay. (D) DNA isolated and purified from ChIP immunoprecipitated material was amplified by PCR with primers to amplify an MDK promoter fragment spanning the SP1 site; equal amounts of total genomic DNA were used for input. Data are representative of three separate experiments.

    Journal: Molecular Biology of the Cell

    Article Title: Transcriptional factor specificity protein 1 (SP1) promotes the proliferation of glioma cells by up-regulating midkine (MDK)

    doi: 10.1091/mbc.E14-10-1443

    Figure Lengend Snippet: An SP1 site located at the MDK promoter is necessary for MDK promoter activation in glioma cell lines. ( A) SP1-knockdown stable U87 and U251 cell lines were transfected with MDK (-562/+33)-luc or pGL-3 Bacis vector and pRL-TK. 24 h after transfection, the levels of luciferase activity were measured and normalized to Renilla luciferase activity. (B) SP1 overexpression and control A172 cell lines were transfected with MDK (−562/+33)-luc or pGL-3 Bacis vector and pRL-TK. At 24 h after transfection, the levels of luciferase activity were measured. (C) Structures of MDK 5′ sequential deletion constructs. U87 cells were cotransfected with reporter plasmids and pRL-TK for 24 h. Promoter activity was determined by luciferase assay. (D) DNA isolated and purified from ChIP immunoprecipitated material was amplified by PCR with primers to amplify an MDK promoter fragment spanning the SP1 site; equal amounts of total genomic DNA were used for input. Data are representative of three separate experiments.

    Article Snippet: Luciferase assay Glioma cells were seeded in triplicate in 48-well plates and allowed to settle for 24 h. A 100-ng amount of luciferase reporter plasmid or the control-luciferase plasmid, plus 1 ng of pRL-TK Renilla plasmid (Promega, Madison, WI), was transfected into glioma cells using Lipofectamine 2000 reagent (Life Technologies) according to the manufacturer's recommendation.

    Techniques: Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Over Expression, Construct, Isolation, Purification, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Polymerase Chain Reaction

    miR-4666-3p targets TGFBR1 in CRC cells. (A) Bioinformatics analysis of potential targets of miR-4666-3p; (B) TGFBR1 is predicted to be a novel target of miR-4666-3p; (C) 293T cells were cotransfected with empty pmirGLO Dual-Luciferase reporter plasmids or TGFBR1 3′UTR firefly luciferase reporter plasmids and pRL-TK-luciferase plasmids, together with the miR-329 or anti-miR-4666-3p mimic. After 48 h, firefly luciferase activity was measured and normalized to that of Renilla luciferase; (D) CRC cells were transfected with the NC, miR-4666-3p or anti-miR-4666-3p mimics, and expression of TGFBR1 was detected by Western blotting; (E) The activity of the TGF-β/Smad pathway was measured by the SBE luciferase reporter system in colon cancer cells (presented as P1 cells) with distinct treatment combination; (F) Serial sphere formation with different cells (control, miR-4666-3p, anti-miR-4666-3p stable expression and both miR-4666-3p and TGF-βR1 stable expression cells). The data are shown as one typical result from three independent experiments with similar results or as the mean ± SD of three independent experiments. * P

    Journal: Frontiers in Oncology

    Article Title: miR-4666-3p and miR-329 Synergistically Suppress the Stemness of Colorectal Cancer Cells via Targeting TGF-β/Smad Pathway

    doi: 10.3389/fonc.2019.01251

    Figure Lengend Snippet: miR-4666-3p targets TGFBR1 in CRC cells. (A) Bioinformatics analysis of potential targets of miR-4666-3p; (B) TGFBR1 is predicted to be a novel target of miR-4666-3p; (C) 293T cells were cotransfected with empty pmirGLO Dual-Luciferase reporter plasmids or TGFBR1 3′UTR firefly luciferase reporter plasmids and pRL-TK-luciferase plasmids, together with the miR-329 or anti-miR-4666-3p mimic. After 48 h, firefly luciferase activity was measured and normalized to that of Renilla luciferase; (D) CRC cells were transfected with the NC, miR-4666-3p or anti-miR-4666-3p mimics, and expression of TGFBR1 was detected by Western blotting; (E) The activity of the TGF-β/Smad pathway was measured by the SBE luciferase reporter system in colon cancer cells (presented as P1 cells) with distinct treatment combination; (F) Serial sphere formation with different cells (control, miR-4666-3p, anti-miR-4666-3p stable expression and both miR-4666-3p and TGF-βR1 stable expression cells). The data are shown as one typical result from three independent experiments with similar results or as the mean ± SD of three independent experiments. * P

    Article Snippet: The pRL-TK plasmids (Promega) were cotransfected into 293T cells with the negative control mimic (5′-UUCUCCGAACGUGUCACGUTT-3′), miR-4666-3p mimic (5′- CAUACAAUCUGACAUGUAUUU-3′), anti-miR-4666-3p mimic (5′- AAAUACAUGUCAGAUUGUAUG-3′), miR-329 mimic (5′- AACACACCUGGUUAACCUCUUU-3′), or anti-miR-329 mimic (5′-AAAGAGGUUAACCAGGUGUGUGUU-3′) (GenePharma Tech, Shanghai, China) using Lipofectamine 2000 (Invitrogen).

    Techniques: Luciferase, Activity Assay, Transfection, Expressing, Western Blot

    Low expression of miR-4666-3p in the PKH hi subpopulation and miR-4666-3p's effect on its target IFN-γR1/2. (A) Schematic model of sorting PKHhi/low/neg subpopulations; (B) Differentially expressed miRNA in PKH hi and the rest of the cell population. Red denotes high and green denotes low levels of expression; (C) The expression level of miR-4666-3p in different subpopulations of different colon cancer cell lines; (D) CRC cells were transfected with the NC, miR-4666-3p or anti-miR-4666-3p mimics, and the expression of IFN-γR1 and IFN-γR2 was detected by Western blotting, KD, knockdown; NC, negative control; (E,F) 293T cells were co-transfected with empty pmirGLO Dual-Luciferase reporter plasmids or IFN-γR1 and IFN-γR2 3'UTR firefly luciferase reporter plasmids, pRL-TK-luciferase plasmids, and the miR-4666-3p or anti-miR-4666-3p mimics. After 48 h, firefly luciferase activity was measured and normalized to that of Renilla luciferase. The data are shown as one typical result from three independent experiments with similar results or as the mean ± SD of three independent experiments. p

    Journal: Frontiers in Oncology

    Article Title: miR-4666-3p and miR-329 Synergistically Suppress the Stemness of Colorectal Cancer Cells via Targeting TGF-β/Smad Pathway

    doi: 10.3389/fonc.2019.01251

    Figure Lengend Snippet: Low expression of miR-4666-3p in the PKH hi subpopulation and miR-4666-3p's effect on its target IFN-γR1/2. (A) Schematic model of sorting PKHhi/low/neg subpopulations; (B) Differentially expressed miRNA in PKH hi and the rest of the cell population. Red denotes high and green denotes low levels of expression; (C) The expression level of miR-4666-3p in different subpopulations of different colon cancer cell lines; (D) CRC cells were transfected with the NC, miR-4666-3p or anti-miR-4666-3p mimics, and the expression of IFN-γR1 and IFN-γR2 was detected by Western blotting, KD, knockdown; NC, negative control; (E,F) 293T cells were co-transfected with empty pmirGLO Dual-Luciferase reporter plasmids or IFN-γR1 and IFN-γR2 3'UTR firefly luciferase reporter plasmids, pRL-TK-luciferase plasmids, and the miR-4666-3p or anti-miR-4666-3p mimics. After 48 h, firefly luciferase activity was measured and normalized to that of Renilla luciferase. The data are shown as one typical result from three independent experiments with similar results or as the mean ± SD of three independent experiments. p

    Article Snippet: The pRL-TK plasmids (Promega) were cotransfected into 293T cells with the negative control mimic (5′-UUCUCCGAACGUGUCACGUTT-3′), miR-4666-3p mimic (5′- CAUACAAUCUGACAUGUAUUU-3′), anti-miR-4666-3p mimic (5′- AAAUACAUGUCAGAUUGUAUG-3′), miR-329 mimic (5′- AACACACCUGGUUAACCUCUUU-3′), or anti-miR-329 mimic (5′-AAAGAGGUUAACCAGGUGUGUGUU-3′) (GenePharma Tech, Shanghai, China) using Lipofectamine 2000 (Invitrogen).

    Techniques: Expressing, Transfection, Western Blot, Negative Control, Luciferase, Activity Assay