Pezx fr01

The FBXO9 promoter (bp -1150/+200 relative to the ATG start codon) and two 5’-truncated sequences of the FBXO9 promoter were cloned into the dual-luciferase reporter gene vector pEZX-FR01 (GeneCopoeia). Lipofectamine 2000 (11668030, Invitrogen) was used to transfect the corresponding dual-luciferase reporter along with ZNF143 or the vector plasmid into Li7 and HCC-LY10 cells. Two days later, luciferase activity of the cells was detected using a dual-luciferase reporter gene assay system (E1980, Promega, Madison, WI, USA).

The promoter sequence of Cdh1 was cloned into pEZX-FR01 (GeneCopoeia) before the Firefly luciferase (Fluc), the Renilla luciferase (Rluc) was used as tracer gene. mESCs were planted in 24-well plates at 2 × 10⁴ per well, and the Tfap2c or control vector (0.5 µg) and pEZX-FR01 (100 ng) were cotransfected into the cells with Lipofectamine Stem Transfection Reagent (Invitrogen, STEM00015). At 48 h after transfection, the luciferase activity was detected according to the instructions for the Luc-Pair Duo-Luciferase Assay Kit 2.0 (GeneCopoeia).

Full-length cDNAs of human LYPLAL1-DT (1,629 bp) and CTNNB1 (2,346 bp) were biosynthesized and then cloned into the plasmid vectors pcDNA3.1 and pEZ-Lv201, respectively, for transient and stable transfection (GeneCopoeia, MD, USA). Likewise, full sequences of human FOXO1 (1,968 bp) and hnRNPK (1,392 bp) cDNAs were synthesized and cloned into plasmid pcDNA3.1 (Umine Biotechnology Co., Guangzhou, China). Additionally, the promoter of LYPLAL1-DT was cloned from whole-genome DNA of MDA-MB-231 and subcloned into plasmid pEZX-FR01 (GeneCopoeia, MD, USA). Truncations of hnRNPK were obtained with appropriate primers from PCR and subcloned into pcDNA3.1 with 3X Flag c-tag. The corresponding empty vectors were used as an overexpression control (Scramble, pcDNA3.1-Scramble, or Vector). All abovementioned constructions were validated by sequencing. A mixture of three siRNAs and three antisense oligonucleotides (ASOs) for knocking down LYPLAL1-DT was provided by Ribobio Corporation (Guangzhou, China), and other siRNAs targeting FOXO1 and hnRNPK were biosynthesized by GenePharma Corporation (Shanghai, China), while nontargeting siRNA was employed as a control of knockdown (siCtrl). All the detailed sequences of siRNAs or ASOs above are provided in Table S1. All transfections were finalized with the Lipofectamine 3000 kit (Invitrogen, CA, USA) following the manufacturer’s instructions.