Pgl3 ap 1

An sgRNA plasmid (MITF GFP 2X-sgRNA), which harbors a mitfa:GFP insert as well as a 2X sgRNA cassette (Fig. 2A), was generated for the zebrafish screen. The 2X sgRNA cassette from MAZERATI 2X (MAS2X) sgRNA (RRID Addgene_118844), also called MiniCoopR 2X sgRNA (gift from Dr. Len Zon), was amplified and introduced into mitfa:GFP plasmid using In-fusion cloning. Site-directed mutagenesis was performed to introduce NheI sites spanning sgRNA2 of the plasmid. MAS2X sgpten was cloned from MAS2X sgRNA (Supplementary Fig. S2A; ref. 44, 109 (link)). The U6-sgRNA plasmid (RRID Addgene_64245) was used along with mitfa:Cas9 plasmid and MiniCoopR:tdTomato (previously developed in the lab). LentiCRISPRV2 Puro (RRID Addgene_52961), psPAX2 (RRID Addgene_12260), and MD2 (RRID Addgene_12259) plasmids were obtained from Addgene. TRIPZ shRNA plasmids were purchased from Dharmacon expressing a nontargeting (NT) shRNA or shRNAs against human GRAMD1B. Clone IDs and hairpin sequences are in Supplementary Table S3. Control luciferase plasmid (pGL3, RRID Addgene_48743) and Renilla luciferase plasmid (pRL-SV40, RRID Addgene_27163) were obtained from Promega (E1751 and E2231) and pGL3-AP-1 was from Addgene (RRID Addgene_40342). Details of the oligonucleotide sequences used for cloning and sequencing are in Supplementary Table S3.

The dual-luciferase reporter assay was performed as described previously.20 (link) Briefly, the renilla luciferase reporter construct pRL-SV40 plasmid was co-transfected with the pGL3-Basic control or the pGL3-AP-1 (#40,342, Addgene) construct into human colorectal cancer cells. The compound, piperlongumine, was added to the cell culture medium and maintained for another 24 h. The cell lysates were collected following the standard protocol and subjected to dual reporter assay using the Dual-Luciferase reporter assay kit (#E1910; Promega, Madison, WI, USA).