Neomycin g418

The luciferase-expressing OPGR80 cell line was derived from a transgenic mouse model of conditional osteosarcoma. The cell line was kindly donated by Dr. Carl Walkley (St. Vincent’s Institute, Melbourne, VIC, Australia). In this model, osteoblast-restricted deletion of p53 and pRb generate spontaneous multifocal osteosarcoma (7 (link)). The luciferase-containing vector was kindly provided by Dr. Andrew Kung (Dana-Farber Cancer Institute, Boston, MA, USA). Luciferase and phosphotransferase sequences were fused and introduced into a pMMP retrovirus to generate pMMP-LucNeo. Cells were co-transfected with pMMP-LucNeo and Eco Pac plasmid. Viral supernatants were then applied to the OPGR80 cell line for 48 h. Luciferase-expressing cells were selected with 1 mg/mL neomycin (G418, Invitrogen, Carlsbad, CA, USA).
OPGR80 cells were cultured under standard conditions of 37°C/5% CO₂ in complete medium (CM), which consisted of MEM-Alpha + GlutaMAX (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic–antimycotic (Invitrogen, Carlsbad, CA, USA).

Human renal carcinoma cell lines (Caki-1, Achn, 786-o, 769-p, A498) and normal human renal tubular epithelial cells HKC were obtained from the Cell Type Collection Center of the Institute of Basic Medicine, Chinese academy of medical sciences (Beijing, China). The catalogue numbers of these cell lines are listed in S1 Table. 786-o and 769-p cells were cultured in RPMI 1640 medium (Hyclone) with 10% FBS (Gibco). HKC cells were cultured in DMEM/F12 (HyClone) medium with 10% FBS, added with 100 U/ml penicillin and 100 U/ml streptomycin (Invitrogen). Caki-1 cells were cultured in HAM’S/F-12 medium with 10% FBS, Caki-1-con and Caki-1-IMP3 were grown in above medium with 5μg/ml Blasticidin (Invivogen). Achn and A498 cells were cultured in minimum essential medium (MEM; HyClone) with 10% FBS, Achn-con shRNA and Achn-IMP3shRNA cells were grown in above medium with 300 μg/ml neomycin G418 (Invitrogen). Growth media were changed every other day. Mouse anti-β-actin antibody was bought from Zhongshan (Guangzhou, China). Rabbit anti-P50, P52, P65 and IκB-α phosphorylated at S32+S36 were purchased from Bioworld (Nanjing, China). NF-κB pathway inhibitor (BAY 11–7082) was bought from Sigma (Sigma-Aldrich).

A PiggyBac vector with Neomycin resistance gene and an inducible iMC-linker Halo sequence (JHL9-pPB-tetON-lynlinker-Halo-CH(utr)_optm) was co-nucleofected with a PiggyBac transposase plasmid at volume proportions of 1:2. Nucleofection was performed using Lonza nucleofector kit V (VVCA- 1003) and the Lonza-AmaxaII device with program A-32 following manufacturer instructions. 72 h after nucleofection, successfully nucleofected PDO were selected with 400 μg ml⁻¹ neomycin (G418, Invitrogen). Vector expression was induced 16 h before experiments by adding 1 μg ml⁻¹ Doxycycline. iMC-linker was labeled using HaloTag Oregon Green Ligand (Promega). Labeled cells expressing the linker were selected and sorted FACS Aria flow cytometer (BD Bioscience).

Each retroviral vector and the pLC 10A1 retroviral packaging vector (Imgenex, San Diego, CA, USA) was co-transfected into HEK293T cells using the Lipofectamine LTX reagent (Invitrogen). After 24 h, the conditioned medium was collected as a viral solution. The retroviral vectors were infected into the cells in the medium that contained 10 μg/mL polybrene (Sigma, St. Louis, MO, USA) and allowed to incubate for 24 h. Then, the viable cells were selected using 800 μg/mL neomycin (G418; Invitrogen). The selected pooled clones were used in the biological analyses. The transfection efficiency was determined using qPCR analysis.

3T3-L1 preadipocytes (2 × 10⁶ cells) were nucleofected with each construct (0.1, 0.5, 1, and 2 ug DNA) using the Amaxa Cell Line Nucleofector Kit V (Lonza #VCA-1003). Each nucleofection was seeded into a 6 well microtiter plate in calf serum medium and expanded for 6 days with700 ug of neomycin G418 (Invitrogen #10131035). Cells were transferred to 50 mm petri plates and grown. Two days after reaching confluence, cells were induced to differentiate for two days with DMEM media containing 10 % fetal bovine serum (FBS) (Life Technologies, #10437028), 0.5 μM 3-isobutyl-1-methylxanthine (IBMX, Sigma #I5879), 1 μM dexamethasone (Sigma #D4902) and 167 nM insulin (Sigma #I-6634). Cells were maintained for the next two days in DMEM medium containing 10 % FBS and 167 nM insulin. Cells were cultured for an additional 4 days in 10 % FBS/DMEM medium, during which time they differentiated into MAs filled with lipid-droplets. MAs were maintained in 10 % FBS/DMEM medium and analyzed daily for green or red fluorescent protein expression by fluorescence microscopy (FM) on Nikon (Eclipse TE2000-S) inverted microscope.