luciferase reporter plasmids prl-sv40  (Promega)

Journal: Cell Death Discovery

Article Title: The EIF4EBP1 gene encoding 4EBP1 is transcriptionally upregulated by MYC and linked to shorter survival in medulloblastoma

doi: 10.1038/s41420-025-02601-x

Figure Lengend Snippet: A ChIP peak locations within the human EIF4EBP1 promoter, exon 1 and part of intron 1 (hg38; Chr8: 38,030,342 - 38,031,906) from ChIP-sequencing data for MYC (Encode consortium, Encyclopedia of DNA Elements at UCSC [ , ]) and an illustration of the luciferase reporter construct containing the EIF4EBP1 promoter, exon 1 and part of intron 1 (−192; +1372) coupled to Firefly luciferase, with the indicated binding sites of the transcription factor MYC. The three E boxes present in the promoter, and corresponding introduced mutations, are indicated. B , C HEK293-T cells were transfected with the (−192; +1372) EIF4EBP1 promoter reporter construct, together with 25 ng, 50 ng and 100 ng MYC ( B ) or the (−192; +1372) EIF4EBP1 promoter reporter constructs containing a mutation of each of the E boxes (as indicated in A ), together with 25 ng MYC ( C ). For ( B ) and ( C ), a Renilla Luciferase vector was used as an internal control and luciferase activities were detected using the Dual-Luciferase Reporter Assay. Firefly luciferase activity was normalized to Renilla luciferase activity and the ratio was normalized to the corresponding 0 ng ( B ) or control ( C ) condition. Data represent the mean of four ( B ) or three ( C ) independent replicates ± standard deviation (SD). Significance was calculated using an unpaired and two-tailed parametric t test (* p < 0.05, **** p < 0.0001). A representative immunoblot analyzing expression of MYC is presented in ( B ). D , E Med8A ( D ) and HD-MB03 ( E ) MB cells were transiently transfected with negative control siRNAs (siCtrl), or two different siRNAs each targeting MYC (siMYC#1 and siMYC#2). Cells were re-transfected after 96 h with their corresponding siRNA and incubated for a total of 168 h. MRNA was harvested to determine the expression levels of EIF4EBP1 and MYC by qRT-PCR. Data obtained by qRT-PCR represent the mean of three independent replicates ± SD and the fold change in expression was normalized to the negative control (siCtrl). Significance was calculated using an unpaired and two-tailed parametric t test (* p < 0.05, ** p < 0.01, **** p < 0.0001). F Med8A MB cells were transiently transfected with negative control (siCtrl) or a pool of four different siRNAs (see Table ) targeting MYC (siMYC). Cells were incubated 72 h and protein was harvested for immunoblotting using the indicated antibodies. G , H Control and MYC overexpressing (MYC OE) ONS76 ( G ) or UW228.3 ( H ) cells were lysed. Levels of EIF4EBP1 mRNA were determined by qRT-PCR. Levels of 4EBP1 and MYC proteins were determined by immunoblots using the indicated antibodies. Data obtained by qRT-PCR represent the mean of three independent replicates ± SD and the fold change in expression was normalized to the control. Significance was calculated using an unpaired and two-tailed parametric t test (*** p < 0.001, **** p < 0.0001).

Article Snippet: Cells were transfected with 125 ng of the EIF4EBP1 promoter Firefly luciferase plasmid (wild type or mutants), 2 ng of Renilla luciferase expressing pRL SV40 plasmid (E2231, Promega; Madison, Wisconsin; USA), as internal control, and 25 ng, 50 ng or 100 ng of MYC expressing pcDNA3.3 MYC plasmid (kindly provided by Dr. Nan Qin, Düsseldorf, Germany), completed to 500 ng total DNA with pcDNA3.1 plasmid (V79020, Thermo Fisher Scientific) using CalFectin TM Cell Transfection Reagent (SL100478, SignaGen Laboratories, Frederick, MD; USA) according to the manufacturer’s guidelines.

Techniques: ChIP-sequencing, Luciferase, Construct, Binding Assay, Transfection, Mutagenesis, Plasmid Preparation, Control, Reporter Assay, Activity Assay, Standard Deviation, Two Tailed Test, Western Blot, Expressing, Negative Control, Incubation, Quantitative RT-PCR