Pcmv gag pol

To generate SNX1 rescue and SNX5 rescue HT1080 cells, retrovirus particles were prepared by transfecting HEK293T cells with pQCXIP-HA-SNX1 (WT, BT*, SAH* or BT-SAH*) or pQCXIP-HA-SNX5 (WT, BT*, SAH* or BT-SAH*) and retrovirus-packaging plasmids pCMV-Gag-Pol (Cell Biolabs, RV-111) and pCMV-VSV-G (Cell Biolabs, RV-110) using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015) according to the manufacturer’s instructions. Medium was collected 24 h after transfection and centrifuged for 10 min at 1,000 × g to remove debris. The double SNX1-2 KO or double SNX5-6 KO HT1080 cells were immediately infected with the corresponding virus, and stably transduced cells were selected with 2 μg ml⁻¹ puromycin.

Stable cells were generated by retroviral transduction through subcloning into the MSCV-PIG vector (Addgene Plasmid #18751). Synthetic complementary DNA corresponding to human NDUFB4 (NM_004547.6), or modified with N24A and R30A substitutions were purchased from GenScript. Gibson assembly (New England Biolabs, E5510S) was performed to ligate complementary DNA constructs to XhoI/HPAI restriction digested and agarose gel purified MSCV-PIG vector. Resulting bacterial clones were verified to contain the insert using Sanger sequencing with the following primer: 5′-CCCTTGAASSTCCTCGTTCG-3′. Bacterial clones were amplified using the PureLink HiPure Plasmid Midiprep Kit (Invitrogen). Packaging plasmids pCMV-VSVG and pCMV-Gag-Pol (Cell biolabs, inc), and the backbone constructs were cotransfected into unmodified HEK293T cells using Lipofectamine LTX reagent (Thermo Fisher Scientific) in a 1:2:3 ratio. Forty-eight hours posttransfection, viral supernatant was harvested and filtered through a 0.45 μm filter. One milliliter of viral supernatant was combined with 400,000 NDUFB4-KO cells and 8 μg/ml hexadimethrine bromide (polybrene) and centrifuged at 800g for 1 h. Cells were then incubated with viral supernatant for 48 h, followed by replacement media supplemented with 2 μg/ml puromycin. To increase expression, cell lines required an additional round of retroviral transduction.

Single guide RNAs (sgRNAs, Supplementary Table 2) were designed using the VBC score sgRNA prediction tool^{82 (link)} and cloned into a lentiviral vector enabling expression of the sgRNA and IRFP670 (pLenti-hU6-sgRNA-PGK-IRFP670). The ABCC1 overexpression construct was a gift from Scott Dixon^{83 (link)}. For the reconstitution experiment, silent point mutations were introduced into the ABCC1 cDNA using PCR mutagenesis to avoid targeting by ABCC1 sgRNAs and the product was cloned into a pMSCV-PGK-BlastR retroviral expression vector using Gateway cloning. The psPAX2 (Addgene plasmid #12260) and pMD2.G (Addgene plasmid #12259) were gifts from Didier Trono. pCMV-gag/pol was acquired from Cell Biolabs, San Diego, USA.