p gl3  (Addgene inc)

Journal: International Journal of Molecular Sciences

Article Title: Hepatocyte Nuclear Factor 4α (HNF4α) Plays a Controlling Role in Expression of the Retinoic Acid Receptor β ( RARβ ) Gene in Hepatocytes

doi: 10.3390/ijms24108608

Figure Lengend Snippet: HNF4α regulates the promoter of the RARβ gene in HepG2 cells. HepG2 cells in 24-well plates were co-transfected for 24 h with a fragment of the human RARβ promoter expanding from −1.7 kbp from transcription start site to +0.217 kpb, as the full length promoter construct, in p GL3-basic-luc vector together with p RLTK plasmid containing Renilla-luc, as the control, and with either human HNF4α, RARα/RXRα, or all three transcription factors, and then treated further either without ( A ) or with RA for 1 to 24 h, after which the cells were collected to assay for luciferase activity. HNF4α alone or with RARα/RXRα suppressed the promoter activity of the human RARβ gene in the cells treated without RA ( A ), but upregulated the promoter when the cells were treated with RA following transfection ( B ). HNF4α regulates the promoter of the mouse RARβ gene in HepG2 cells ( C ). It suppresses the promoter activity of the mouse gene (empty bars in C) in HepG2 cells treated with the vehicle, but it increases the promoter activity in the cells treated with RA (black bar in C).

Article Snippet: Construction of the plasmid vectors including p GL3-Basic-hCYP26A1-E4-luc (submitted to addgene.org), p GL3-Basic-hRARβp-luc (human RARβ2 promoter), p GL3-Basic-hCYP2C9p-luc, p cDNNA3.1-hRARα.hRXRα (submitted to addgene.org), and p cDNA3.1-hHNF4α were reported previously [ , , , , ].

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

Journal: International Journal of Molecular Sciences

Article Title: Hepatocyte Nuclear Factor 4α (HNF4α) Plays a Controlling Role in Expression of the Retinoic Acid Receptor β ( RARβ ) Gene in Hepatocytes

doi: 10.3390/ijms24108608

Figure Lengend Snippet: Mutation of the critical residues present in the ligand binding domain of human HNF4α suppresses transcription activation of the promoters of human APOC3 and CYP2C9 genes. HepG2 cells were co-transfected with either the p GL3-b-hApoC3 ( A – C ) or p GL3-b-hCYP2C9 ( D – F ) promoter construct together with either hHNF4α, RARα/RXRα, or all three transcription factors, and then treated with either vehicle or 1 µM RA for 24 h, after which the cells were assayed for their luciferase activity. The effects of the individual mutant residues in the ligand binding domain of the human HNF4α compared with WT HNF4α on the promoter activity of APOC3 ( B ) or CYP2C9 ( E ) in HepG2 cells treated with either vehicle or 1 μM RA for 24 h. The endogenous HNF4α transcriptional activity toward APOC3 ( C ) and CYP2C9 ( F ) promoters was assessed in HepG2 cells with or without the addition of human HNF4α mutants. Data from each bar represent the mean of n = 3 wells ± SD. HNF4α mutant #’s are as follows: (1) S190K, (2) 191MK, (3) R221G, (4) L228K, (5) L229K, (6) R235G, (7) I347K, and (8) I355K.

Article Snippet: Construction of the plasmid vectors including p GL3-Basic-hCYP26A1-E4-luc (submitted to addgene.org), p GL3-Basic-hRARβp-luc (human RARβ2 promoter), p GL3-Basic-hCYP2C9p-luc, p cDNNA3.1-hRARα.hRXRα (submitted to addgene.org), and p cDNA3.1-hHNF4α were reported previously [ , , , , ].

Techniques: Mutagenesis, Ligand Binding Assay, Activation Assay, Transfection, Construct, Luciferase, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Hepatocyte Nuclear Factor 4α (HNF4α) Plays a Controlling Role in Expression of the Retinoic Acid Receptor β ( RARβ ) Gene in Hepatocytes

doi: 10.3390/ijms24108608

Figure Lengend Snippet: Mutation of the critical amino acid residues present in the ligand binding domain of human HNF4α suppresses transcription activation of the promoters of human CYP26A1 and RARβ genes in HepG2 cells treated with RA. HepG2 cells grown in 24-well plates were co-transfected with either p GL3-b-hCYP26A1 ( A ) or p GL3-b-hRARβ ( B ) promoters, each with p RLTK as the control, together with either wildtype (WT) HNF4α or its individual mutants (Mutant # 1 to 8), and then treated with either vehicle or 1 µM RA for 24 h, after which the cells were assayed for luciferase activity. Data from each bar represent the mean of n = 3 wells ± SD. HNF4α mutant #’s are as follows: (1) S190K, (2) 191MK, (3) R221G, (4) L228K, (5) L229K, (6) R235G, (7) I347K, and (8) I355K.

Article Snippet: Construction of the plasmid vectors including p GL3-Basic-hCYP26A1-E4-luc (submitted to addgene.org), p GL3-Basic-hRARβp-luc (human RARβ2 promoter), p GL3-Basic-hCYP2C9p-luc, p cDNNA3.1-hRARα.hRXRα (submitted to addgene.org), and p cDNA3.1-hHNF4α were reported previously [ , , , , ].

Techniques: Mutagenesis, Ligand Binding Assay, Activation Assay, Transfection, Control, Luciferase, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Biphasic transcriptional and posttranscriptional regulation of MYB by androgen signaling mediates its growth control in prostate cancer

doi: 10.1016/j.jbc.2022.102725

Figure Lengend Snippet: Effect of miR-150 inhibition and restoration on MYB expression in low- and high-dose DHT-treated prostate cancer cells. A and B , LNCaP and C4-2 cells were transfected with negative control-inhibitor (NC-Inh) or miR-150 inhibitor (miR150-Inh) for 24 h, followed by low and high doses treatment of DHT for another 24 h. MYB expression was analyzed by Western blotting. β-actin was used as a loading control. C and D , cells were transfected with negative control miRNA mimic (miR-NC) or miR-150 mimic (miR-150) for 24 h, followed by DHT (1.0 nM) treatment for 24 h and MYB expression analysis. E – G , prostate cancer cells were transfected with a luciferase reporter plasmid carrying MYB-3ʹUTR-WT (WT) sequence containing a putative-binding site for miR-150 or MYB-3ʹUTR-Mut (Mutant) where miR-150 binding site is mutated. Cells were also cotransfected with Renilla luciferase plasmid (pRL/TK) to control transfection efficiency. After 24 h, cells received treatment with low and high doses of DHT, and luciferase activity was examined after 24 h of incubation. The data is presented as normalized luciferase activity (mean ± SD), ∗ p = < 0.05. H and I , LNCaP and C4-2 cells were cotransfected with pGL3-MYB-3′UTR WT or pGL3-MYB-3′UTR mutant (Mut) luciferase along with either control-inhibitor (NC-Inh) or miR-150 inhibitor (miR150-Inh) for 24 h. Thereafter, cells were treated with low and high DHT doses for 24 h. Luciferase activity was examined, and the data presented as the normalized luciferase activity (mean ± SD), ∗ p = < 0.05, n.s. not significant. DHT, dihydrotestosterone.

Article Snippet: WST-1 reagent was purchased from BioVision, Inc. MYB promoter-reporter construct (GLuc-ON MYB; HPRM34981-PG02), MYB mutant promoter-reporter construct (GLuc-ON MYB; HPRM34981-PG02-01), has-miR-150 WT and mutant (CS-HPRM54623-PG04-01 and CS-HPRM54623-PG04-02) were purchased from GeneCopoeia. p-GL3-MYB-3′UTR (Cat. # 25798) (Firefly Luciferase) ( ) was purchased from Addgene, p-GL3-MYB-3′ UTR-mutant (VB20819-12933men) was purchased from VectorBuilder, and pRL-Tk (Renilla Luciferase) plasmid constructs was purchased from Promega.

Techniques: Inhibition, Expressing, Transfection, Negative Control, Western Blot, Luciferase, Plasmid Preparation, Sequencing, Binding Assay, Mutagenesis, Activity Assay, Incubation

Journal: Bioactive Materials

Article Title: Combining mannose receptor mediated nanovaccines and gene regulated PD-L1 blockade for boosting cancer immunotherapy

doi: 10.1016/j.bioactmat.2021.05.036

Figure Lengend Snippet: Characterization of HA/PLL-RT/pDNA nanoparticles (NPs). Zeta potential (A), Particle size (B), SEM (C), and stability of NPs in FBS (D) at different incubation time (0 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h).

Article Snippet: Luciferase plasmid DNA ( p GL3), luciferin substrate, and cell lysis buffer were purchased from Promega (Madison, USA).

Techniques: Zeta Potential Analyzer, Incubation

Journal: Bioactive Materials

Article Title: Combining mannose receptor mediated nanovaccines and gene regulated PD-L1 blockade for boosting cancer immunotherapy

doi: 10.1016/j.bioactmat.2021.05.036

Figure Lengend Snippet: Characterization of cytotoxicity, endocytosis and transfection efficiency of HA/PLL-RT/pDNA nanoparticles in B16F10 cells. (A) Cytotoxicity of pDNA, PLL-RT, HA, PLL-RT/pDNA and HA/PLL-RT/pDNA nanoparticles. (B) Flow cytometry of PLL-RT/FAM-DNA nanoparticles after introducing different amount of HA. (C) Intracellular uptake of PLL-RT/FAM-DNA nanoparticles by CLSM after 4 h incubation. Scale bar = 20 μm. (D) In vitro gene transfection efficiency of HA/PLL-RT/ p GL3-control at various HA contents. (E) PD-L1 mRNA expression after treating with different formulations for 24 h.

Article Snippet: Luciferase plasmid DNA ( p GL3), luciferin substrate, and cell lysis buffer were purchased from Promega (Madison, USA).

Techniques: Transfection, Flow Cytometry, Incubation, In Vitro, Control, Expressing