Shc007

One day before transfection, 4 × 10⁶ 293FT cells were seeded into a 10 cm dish. Cells were transfected with either OGFOD1 shRNAs (Sigma-Aldrich, #SHCLNG-NM_018233) or control shRNA (shLuciferase) (Sigma-Aldrich, #SHC007) in serum-free DMEM containing viral packaging (psPAX2) and envelope (pMD2G) constructs using Lipofectamine 2000 reagent (Thermo Fisher, #11668019). After 18 h post-transfection, the cell culture medium was replaced with 10 mL of fresh medium, and cells were incubated for an additional 48 h. Lentivirus-containing supernatants were harvested via centrifugation at 2000 rpm for 10 min and residual cell debris removed using a 0.45 μm filter (Sartorius, #16555-K). To knock down the expression of OGFOD1 in MDA-MB-231 cells, 1 mL of lentivirus was infected with polybrene to 5 × 10⁵ cells and OGFOD1 mRNA and protein levels were monitored by RT-qPCR and Western blot analysis, respectively. The full western blots can be found at Figures S6–S15.

pGL3-p21, pGL3-PUMA, pGL3-MDM2 (a gift from Dr David W Meek, University of Dundee, UK), pSV-β-Galactosidase, pCDNA3, pCDNA3-FLAG-MDM2, pCDNA3-GFP-BTK (OriGene, #RG211582), pGEX-MDM2, pGEX-UBC9, shBTK (Santa Cruz sc-29841-sh), and shRNA against luciferase (Sigma Aldrich SHC007, Mission pLKO.1puro Luciferase) were used in this study.

HEK 293T cells were used to produce lentiviral particles containing shTWIST1 (Sigma-Aldrich, TRCN0000020543) and shLuciferase (Sigma-Aldrich, SHC007) (as a negative control). HEK cells (5 × 10⁵ cells per well) were transfected with 5 µg of DNA (per well)—2.38 µg of packaging plasmid pCMV-dR8.74psPAX2, 0.24 µg of envelope plasmid VSV-G/pMD2.G, and 2.38 µg of the shRNA plasmids. Cells were then incubated for 18 h after which medium was replaced to remove the transfection reagent and the cells were incubated for an extra 30 h. The media containing the lentiviral particles were harvested, mixed with PEG-it Virus Precipitation Solution (System Biosciences, LV810A-1) and left overnight at 4 °C. The harvested viral particles were then used to transduce CFBE wt- and F508del-CFTR cells. CFBE cells were infected with 1.5 mL of lentivirus-containing medium. The plates were centrifuged at 200×g for 1 h at 25 °C and then incubated for 24 h at 37 °C, 5% CO₂. The medium was then changed to the respective cell medium supplemented with selection antibiotics to eliminate the non-transduced cells.

Lentiviral constructs encoding small hairpin RNA sequences target- ing NUMB were purchased from GE Dharmacon (Lafayette, CO) (oligo identifications: TRCN 0000007227 and TRCN0000007226, referred to as shNUMB_1 and shNUMB_2, respectively). A lentiviral construct encoding small hairpin RNA sequences targeting lucif- erase (SHC007, Sigma-Aldrich, St. Louis, MO) served as vector control.