Memerald tomm20 n 10

The following GFP-labeled constructs were utilized: Plasma membrane label Src-myrisylated-GFP, pmyr GFP (Addgene plasmid # 50528) was a gift from Kenneth Yamada. Golgi label beta-1,4-galactosyltransferase 1-GFP, PA-GFP (Addgene plasmid # 57164), lysosome Label Lamp-1-GFP, Emerald-lysosome-20 (Addgene plasmid # 56476). ER label SigPep-eGFP-KDEL, mEGFP-endoplasmic reticulum (Addgene plasmid # 56455), mitochondria label mitochondrial import receptor subunit translocase of outer membrane 20 kDa subunit-GFP, mEmerald-TOMM20-N-10 (Addgene plasmid # 54282), nucleus label SV40 NLS-GFP, mEmerald-nucleus 7 (Addgene plasmid # 54206), and cytoplasm label Argonaut 3 isoform A-GFP, mEmerald-EIF2C3-C18 (Addgene plasmid # 54078) were gifts from Michael Davidson. Golgi label Tyrosyl protein sulfotransferase 2, TPST2-EGFP was a gift from David Stephens (Addgene plasmid # 66618). Selections were performed in G418 or by limiting dilution.

SiR-CA was synthesized according to previously reported methods. BacMam 2.0 reagent, CellLight Mitochondria-GFP (PDHA1–GFP; C10600), CellLight Mitochondria-RFP (PDHA1–RFP; C10505), CellLight Golgi-GFP (GALNT1–GFP; C10592), CellLight Golgi-RFP (GALNT1–RFP; C10593), CellLight ER-GFP (KDEL–GFP; C10590), CellLight ER-RFP (KDEL–RFP; C10591), culturing medium DMEM (12430054, 21063029 and A1443001), Opti-MEM with no phenol red (11058021) and FBS and chemical reagents Hoechst 33342 (62249) and MitoTracker Deep Red FM (M22426) were all purchased from Thermo Fisher. CellTiter-Glo 2.0 (G9242) was purchased from Promega, and alexidine dihydrochloride (A8986) was purchased from Sigma-Aldrich. Plasmid encoding mEmerald–TOMM20 was a gift from M. Davidson (Florida State University, mEmerald–TOMM20-N-10, Addgene plasmid 54282). Plasmid encoding Halo-TOMM20 was a gift from K. McGowan (HHMI Janelia, Halo-TOMM20-N-10, Addgene plasmid 123284). Plasmid encoding COX8A-SNAP was a gift from A. Egana (New England Biolabs, pSNAPf-COX8A, Addgene plasmid 101129).

We generated a modified pLKO.1 plasmid backbone with an accessible multiple cloning site (pLKO.1_MCS) for generation of fluorescent reporters. For mito-FP expression, we cloned the cytochrome oxidase subunit VIII mitochondrial targeting sequence and tagged it to mEmerald (referred to as mito-mEm) or tagRFPt (referred to as mito-RFP) and introduced these into the pLKO.1_MCS backbone in order to generate lentiviruses. pLKO.1 mito-mEmerald and pLKO.1 mito-TagRFP-T are available on Addgene (#174542 and 174543, respectively). For FP-TOMM20 expression, inserts containing the sequence of either mEmerald (mEmerald-TOMM20) or mcherry (mCherry-TOMM20) fused to TOMM20 and cloned into the pLKO.1 backbone and used to generate lentiviruses. Stable lines were generated through lentiviral transduction. For transduction, approximately 50,000 cells were plated into one well of a 6-well plate directly into the appropriate lentivirus supernatant diluted 1:5 in DMEM complete media with a final concentration of 10 µg/mL polybrene (TR-1003-G, Sigma). After 48–72 hr, cells were expanded, and multiclonal populations were flow sorted for appropriate levels of fluorescent expression. All other transgenic cell lines were generated as outlined in subsequent sections.
mEmerald-TOMM20-N-10 (Addgene plasmid # 54282) and mCherry-TOMM20-N-10 (Addgene plasmid # 55146) were a gift from Michael Davidson.