Sc 108060

Recombinant lentivirus particles were produced in HEK293T cells by transient transfection of lentivirus packaging plasmids, including 4 plasmids expressing Gag-Pol (pHDM-Hgpm2), Tat (pHDM-tat1b), Rev (pRC-CMV-rev1B), and VSV-G (pHDM VSV-G), and a pool of 3 recombinant lentiviral vectors, each encoding shRNA specific for cPLA2 (Santa Cruz Biotechnology Inc., sc-35098-SH) or scrambled shRNA (Santa Cruz Biotechnology Inc., sc-108060). Lentiviral particles packaged with LPCAT1 overexpressing plasmid were produced by co-transfection of lentivirus packaging plasmids, as described above, with pHAGE-EF1α-3xFlag vector or pHAGE-EF1α-3xFlag-LPCAT1 into HEK293T cells. For construction of pHAGE-EF1α-3xFlag-LPCAT1 plasmid, full length of LPCAT1 coding sequence was inserted into pHAGE-EF1α-3xFlag vector to generate pHAGE-EF1α-3xFlag-LPCAT1 plasmid. Cell culture media containing viruses was collected 3 times within 72 hours after transfection and concentrated by centrifugation62 (link). Concentrated virus stocks had a titer greater than 2 × 10⁸ per ml and were stored at −80 °C before bone marrow cell (BMC) transduction. BMCs isolation and viral transductions were performed by previously described30 (link). The efficiency of lentivirus transduction was assessed by detection of GFP expression in BMCs using flow cytometry.

The TRIB1 expression plasmid was obtained from GenScript (Clone ID: OHu22886) and system biosciences (SBI) by providing custom sequence (Supplementary Methods 1). The doxycycline inducible shRNA was purchased from Dharmacon (SMARTvector Inducible Human TRIB1 mCMV-TurboRFP shRNA). The TRIB1 deletion mutant constructs were a generous gift from Dr. Takuro Nakamura (Division of Carcinogenesis, The Cancer Institute, Tokyo, Japan). TRIB1-W337A-FLAG and TRIB1-W337A-MYC plasmids were generated by mutating the MEK binding site in respective TRIB1 expression plasmids using QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent technologies) according to manufacturer’s instructions. The primer sequence is indicated in supplementary methods 2. Control shRNA plasmid-A (sc-108060) and COP1 shRNA Plasmid (h) (sc-45541-SH) were purchased from Santa Cruz biotechnologies.

The human ABCC10 or ABCG2 gene was inserted into the EcoRI and XbaI sites of pcDNA3.1(+) (Invitrogen, Carlsbad, CA) to make expression vectors, pcDNA3.1(+)/ABCC10 or pcDNA3.1(+)/ABCG2.
To establish ABCC10-overexpressing NSCLC cells, gefitinib-sensitive PC9 and H292 cells were transfected with pcDNA3.1(+)/ABCC10 or empty vector using Lipofectamine^TM 2000 (Invitrogen, Carlsbad, CA, USA). To establish NSCLC cells with ABCC10 knockdown, ABCC10 shRNA plasmid (shABCC10, Santa Cruz Biotechnology, sc-62641-SH) or control plasmid (shMock, Santa Cruz Biotechnology, sc-108060) was introduced into gefitinib-resistant NSCLC cells PC9/GR and H292/GR. Single colonies were identified in culture medium containing G418 (2 mg/mL) and subcultured for further analysis.
To establish the stably transfected LLC-PK1 cells expressing ABCC10 or ABCG2, pcDNA3.1(+)/ABCC10, or pcDNA3.1(+)/ABCG2 was transfected into LLC-PK1 cells, and stable transfected clones were selected as described above. Expression of ABCC10/ABCG2 was confirmed by quantitative real-time PCR and western blot analysis as described below.