Megfp n1

pDsRed2-Mito (MitoDsRed) was a gift of G. Hajnoczky (Thomas Jefferson University, Philadelphia, PA, United States). For shRNA constructs, pLKO.1-non-target shRNAs and pLKO.1-PP2Ac shRNAs (TRCN0000002484) were obtained from Sigma-Aldrich (St. Louis, MI, United States). The EGFP fragment was amplified by PCR from mEGFP.N1 and cloned into pLKO.1-non-target-shRNA (Scrambled) and pLKO.1-hPP2A-shRNA (PP2A shRNA) to generate pLKO.1-non-target-EGFP and pLKO.1-hPP2A-shRNA-EGFP. pLKO.1-non-target-EGFP and pLKO.1-hPP2A-shRNA-EGFP expressing lentivirus was produced at Boston Children’s Hospital (BCH) Viral Core. PP2A-shRNA-Resistant-BFP was generated using Q5 Hot Start High-Fidelity site-directed mutagenesis kit (New England Biolabs, #M0494, Ipswich, MA, United States). mCherry-Rab7 (#55127) and mEGFP-N1 (#54767) were obtained from Addgene.

To generate the pAAVS_Nst_CAG_mEGFP_PTS1 vector, the pENTR_mEGP_PTS1 was generated at first. Initially, the mEGFP-N1 (plasmid #54767; Addgene) was digested by BsrgI/NotI, and a double oligo (top PST1 and bottom PST1, see Table S1) with overhang was used for sticky-end ligation. The new plasmid, called mEGFP-N1-PTS1, was then digested with BamHI and XbaI and the fragment containing the mEGFP_PTS1 sequence was cloned into the pENTR2B_kan_adaptor (gift from Prof. William Skarnes from The Jackson Laboratory, Farmington, CT, USA), previously digested with BamHI and XbaI. Once the sequence of the pENTR_mEGP_PTS1 was confirmed by Sanger sequencing, the plasmid was recombined using LR clonase (Gateway LR Clonase II Enzyme mix, Cat. #11791020; Thermo Fisher Scientific) into the destination vector pAAVS1_Nst_CAG_DEST (plasmid #80489; Addgene).

All DNA constructs were produced using Escherichia coli DH5a (Thermo Fisher Scientific) and extracted using a plasmid midiprep kit from Qiagen. The plasmids used in this study were mEGFP-N1 (plasmid #54767; Addgene); pHyPer-cyto (FP941; Evrogen); and pHyPer-pexo, pHyPer-endo, PEX3_Turbo, and pAAVS_Nst_CAG_mEGFP_PTS1 generated in our lab.

All DNA constructs were produced using Escherichia coli DH5a (Thermo Fisher Scientific) and extracted using a plasmid midiprep kit from Qiagen. The plasmids used in this study were mEGFP-N1 (plasmid #54767; Addgene); pHyPer-cyto (FP941; Evrogen); and pHyPer-pexo, pHyPer-endo, PEX3_Turbo, and pAAVS_Nst_CAG_mEGFP_PTS1 generated in our lab.

The AT-3 tumor cell line was established from a primary mammary gland carcinoma of the PyMT-MMTV transgenic mice on a B6 strain and was maintained as described^{66 (link)}. The 4T1 breast cancer and B16-F10 (B16) melanoma cell lines were purchased from the American Type Culture Collection (ATCC), authenticated at ATCC, and maintained. The MC38 colon adenocarcinoma cell line was gift from Dr. Weiping Zou (University of Michigan). AT-3 cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Sigma), 1% NEAA (Gibco), 2 mM l-glutamine (Gibco), 0.5% penicillin/streptomycin (Gibco), and 55 μM 2-mercaptoethanol (Gibco). 4T1, MC38, and B16 cells were cultured in RPMI (Gibco) supplemented with 10% FBS, 1% NEAA, 2 mM l-glutamine, 0.5% penicillin/streptomycin, and 55 μM 2-mercaptoethanol. For generation of mouse AT-3 cells expressing GFP (AT-3-GFP), AT-3 cells were infected with retroviruses encoding GFP (mEGFP-N1, Addgene plasmid #54767 a gift from Dr. Michael Davidson). 4T1 cell expressing Luciferase (4T1-luc) were generated with infection of lentiviruses encoding Luciferase (pLenti PGK V5-LUC Neo, Addgene plasmid #21471). These cell lines were authenticated by morphology, phenotype, and growth, and routinely screened for Mycoplasma, and were maintained at 37 °C in a humidified 5% CO₂ atmosphere.