E2940

HEK293T cells (2.5 × 10⁵ cells ml⁻¹) were used to seed a 96-well plate. They were transfected, in triplicate, the following day, with WT and mutant RIPK3 plasmids along with 100 ng NF-κB promoter-firefly luciferase reporter plasmid and 100 ng of Renilla luciferase reporter plasmid per well, in the presence of X-tremegen 9 DNA Transfection Reagent (Roche). Luciferase activities were assessed 24 h later, in a dual luciferase assay (Promega, E2940). Firefly luciferase activities were normalized against Renilla luciferase activities.

Follow our previous method [32 ]. Firstly, construction of reporter gene plasmid. The mouse cyclin D1 promoter sequence was cloned into pGL3-basic plasmid vector to obtain pF1, pF2, pF3, pF4, pF5, and pF6 plasmid. Mutated pF2 was generated by using the Mutant Best Kit (Takara, China). Secondly, transfect cells. At 48 h after pAdEasy-Foxg1 or pAdEasy-Myc transfection, the cells were transfected with the above luciferase reporter expression vectors using Lipofectamine 2000 for the promoter assay, respectively. And then, we used a multi-functional microplate reader to detect the expression level of the reporter gene according to the manufacturer’s instructions (Promega, E2940).

The TargetScan (http://targetscan.org) and miRWalk (Home – miRWalk (uni‐heidelberg.de) were used to identify the putative target genes of miR‐669a‐5p. The sequence of mouse G3BP1 (NM_013716; ENSMUSG00000018583.5) and G3BP2 (NM_001080794; ENSMUSG00000029405.16) were obtained from the GenBank and UCSC Genome Browser. Information on miR‐669a‐5p (MI0004523) was acquired from miRBase.
To construct the luciferase reporters, a part of G3BP1 CDS or 3′UTR and G3BP2 3′UTR containing the miR‐669a‐5p potential target sites was cloned in the pmirGLO Dual‐Luciferase Vector (E1330, Promega). The Mut Express II Fast Mutagenesis Kit V2 (C214‐01, Vazyme) was used for target sequence mutagenesis. The primers for constructing plasmids were listed in Table S5 (Supporting Information). Briefly, cells were co‐transfected with the dual‐luciferase plasmid containing G3BP1 CDS or 3′UTR, G3BP2 3′UTR, or the respective mutated sequences, miR‐669a‐5p mimic (MIC669a), or nontargeting NC (NC_MIC) oligos and were them cultured for 48 h before the luciferase assay. Firefly and Renilla luciferase activities were detected with a dual‐luciferase reporter assay system (E2940, Promega), and the results were shown as firefly luciferase activity relative to Renilla luciferase activity.