Pcdna3.1 p53 plasmid

DACH1 shRNA, p53 shRNA, and SLC25A37 shRNA were obtained from Santa Cruz Biotechnology. The eukaryotic expression vector3 (pcDNA).1-DACH1 plasmid, pcDNA3.1-P53 plasmid, and pcDNA3.1-SLC25A37 plasmid were purchased from Hanbio (Shanghai, China). P53 plasmids with different domains were cloned and inserted into the pcDNA 3.1-myc vector. VA-Lip-Control-shRNA, VA-Lip-SLC25A37-shRNA, VA-Lip-P53-shRNA, and VA-Lip-DACH1-shRNA were prepared according to previous reports.^{8 (link)–10 (link)} According to the manufacturer’s protocols, shRNA and plasmids were transfected by Lipofectamine 3000. Stable clones were selected in 2 µg/mL puromycin for 4 weeks, and a single clone was isolated using a limited dilution technique.

BRD7 shRNA (sc-92998, sc-141741), p53 shRNA (sc-44218, sc-45917), SLC25A28 shRNA (sc-90800, sc-149448), and control shRNA were obtained from Santa Cruz Biotechnology. The pcDNA3.1-BRD7 plasmid (NM_001108440.1, NM_ 013263.5), pcDNA3.1-P53 plasmid (NM_030989.3, NM_000546.5), pcDNA3.1-SLC25A28 plasmid (NM_00110 9515.1, NM_031212.4) and control pcDNA3.1 vector were purchased from Hanbio (Shanghai, China). The different p53 domains were constructed by PCR and cloned into pcDNA3.1-myc vector. The resulting plasmid was verified by sequencing. Transfections were performed with Lipofectamine™ 3000 (Invitrogen, L3000008) according to the manufacturer's instructions. VA-Lip-BRD7-shRNA, VA-Lip-P53-shRNA, VA-Lip-SLC25A28-shRNA and VA-Lip-Control-shRNA were prepared according to our previous reports [12 ,13 (link)]. In brief, 5 mg VA was added into 50 μl DMSO to form VA solution. 280 nmol VA solution and 0.14 μmol lipotrust solution (Hokkaido System Science, LEO-01) were mixed by vortexing in a 1.5-ml tube at 25 °C. 12.24 nmol BRD7 shRNA, p53 shRNA, SLC25A28 shRNA or control shRNA was added into VA-Lip solution with stirring at 25 °C. The VA-Lip solution was filtered. Fractions were collected and the material trapped in the filter was reconstituted with PBS to achieve the desired dose for in vivo use.