Plasmid-Mediated Cell Tracking and Manipulation

Plasmids for tracking PI species were from Addgene. Specifically, mCherry-Clathrin LC-15 (Addgene #55019) [71 (link)]; PLCD1(PH)-mCherry (Addgene #36075) [72 (link)]; PH-PLCD1-GFP (Addgene #51407), PH-PLCD1(R40L)-GFP (Addgene #51408) [73 (link)]; pLentiLifeACT-EGFP BlastR (Addgene #84383) [74 (link)]; PH-Btk-GFP (Addgene #51463), GFP-PH-TAPP1 (Addgene #161985); and pLenti-EGFP-P4M-SidMx2 (Addgene #136997), pLenti-EGFP-2xFYVE (Addgene #136996) were gifts from Ken-Ichi Takemaru. Plasmids were also constructed by Vectorbuilder: VB200917 is a lenti-mCherry-shCLTC containing U6-driven shRNA sequence 5′-CGTGTTCTTGTAACCTTTATT-3′. VB200917 is a lenti-mCherry-shLuciferase containing U6-driven shRNA sequence 5′- ATGTTTACTACACTCGGATAT-3′ and was used to create lentivirus to infect MCF7, 4226, and MPE600 cell lines. Infected cells were sorted for mCherry using FACS as above and tested for engulfment. Human PIK3C2B-GFP fusion overexpression vector is a lentiviral CMV-driven plasmid from VectorBuilder. miRFP-713 (near-infrared fluorescent protein) overexpression vector is a lentiviral EFS-driven plasmid from VectorBuilder. 2xFYVE-domain-mCherry fusion protein is lentiviral plasmid from VectorBuilder. Lentiviral EFS-driven LYN11 mCherry fusion protein construct was also produced by VectorBuilder.