prl c myc 3  (Addgene inc)

Journal: iScience

Article Title: c-JUN inhibits mTORC2 and glucose uptake to promote self-renewal and obesity

doi: 10.1016/j.isci.2022.104325

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Article Snippet: pRL c-Myc 3′UTR , Addgene , Plasmid #14806.

Techniques: FLAG-tag, Recombinant, Protease Inhibitor, Modification, Membrane, Reverse Transcription, SYBR Green Assay, Western Blot, Gel Extraction, Mutagenesis, Reporter Assay, Isolation, cDNA Synthesis, Northern Blot, Plasmid Preparation, shRNA, Software

Journal: The EMBO Journal

Article Title: Smg6/Est1 licenses embryonic stem cell differentiation via nonsense-mediated mRNA decay

doi: 10.15252/embj.201489947

Figure Lengend Snippet: A Heat map showing gene expression profiles from control and Smg6 Δ/Δ ESCs (two of each genotype). A total of 2,449 DEGs were subjected to this analysis. Red and green colors depict high and low gene expression levels, respectively, based on the log 2 P -values of their RPKMs. B All these DEGs were subjected to GO analysis using DAVID. An enrichment map was constructed using Cytoscape installed with the Enrichment Map plug-in. Each enriched GO pathway is represented by a red node ( P < 0.05; FDR < 0.05; overlap cutoff > 0.5). Note that the size of nodes is proportional to the total number of genes in each pathway. Edge thickness represents the number of overlapping genes between nodes. GO pathways of similar function are sorted into one cluster, marked with a circle and label. Gene number in each cluster is in parentheses. C Relative c-Myc mRNA level in control (F/F) and Smg6 Δ/Δ (Δ/Δ) ESCs by qRT–PCR analysis. n : number of ESC lines analyzed. Unpaired Student's t -test was used for the statistical analysis. * P < 0.05. D Western blotting of c-Myc protein expression in Smg6 Δ/Δ ESCs reconstituted with different Smg6 truncations. Note, only NMD-proficient constructs (ΔN-Smg6 and FL-Smg6) could repress the c-Myc protein expression. E Expression of c-Myc isoforms (schemes is shown on the left panel) in control (F/F) and Smg6 Δ/Δ (Δ/Δ) ESCs. The expression level is shown with FPKM calculated with RNA-seq data ( n = 2 for each genotype) (right panel). *** P < 0.001. F Expression of c-Myc protein isoforms in control (F/F) and Smg6 Δ/Δ (Δ/Δ) ESCs. G c-Myc 3′-UTR is a NMD target. A luciferase reporter fused with the c-Myc 3′-UTR was used to determine the mRNA stability in control (F/F) and Smg6 Δ/Δ (Δ/Δ) ESCs. The data are summarized from three independent experiments. * P < 0.05. H Overexpression of c-Myc in wild-type (E14.1) ESCs blocks differentiation. c-Myc overexpressed EBs (day 5) retained a high number of cells expressing Oct4. The percentage of Oct4 + cells in EBs is shown on the right panel. n , the number of cells scored. ** P < 0.01. Unpaired Student's t -test was used for the statistical analysis. I AP staining (left) and Oct4 immunostaining (right) of the day 5 culture of control and Smg6 Δ/Δ ESCs in the spontaneous differentiation assay. J Western blot analysis of Oct4 in c-Myc siRNA-treated Smg6 Δ/Δ ESCs at day 7 in spontaneous differentiation assay. Non-targeted siRNA (NT-siRNA) is a control treatment. Data information: The error bars represent the SEM.Source data are available online for this figure.

Article Snippet: To determine the 3′-UTR in c-Myc mRNA stability, a luciferase reporter plasmid (pRL-c-Myc 3′-UTR, Addgene number: 14806, a kind gift from Tyler Jacks) was co-transfected with a firefly luciferase reporter plasmid (pGL3) into ESCs (Kumar et al , ).

Techniques: Gene Expression, Control, Construct, Quantitative RT-PCR, Western Blot, Expressing, RNA Sequencing, Luciferase, Over Expression, Staining, Immunostaining, Differentiation Assay