Powerplex 1.2 kit

Human pancreatic cancer cell lines AsPC-1, Panc-1, Capan-1, and Mia PaCa-2 were obtained from ATCC (Manassas, VA), the murine cell line Pan02 was obtained from the DCTD tumor repository maintained by the NCI at Frederick. C5LM2 is a variant of Panc1 developed in our laboratory that was generated through 2 passages of growth in vivo and culture of liver metastases and has been characterized previously (11 (link)). C5LM2, AsPC-1, Panc-1, Pan02, and Mia PaCa-2 lines were grown in DMEM, Capan-1 in was grown in IMDM, all cell lines were grown in a humidified atmosphere with 5% CO2, at 37°C, and have been DNA fingerprinted for provenance using the Power-Plex 1.2 kit (Promega) and confirmed to be the same as the DNA fingerprint library maintained by ATCC and were confirmed to be free of mycoplasma (e-Myco kit, Boca Scientific).

Human NSCLC cell lines and immortalized human bronchial epithelial HBEC3KT cells (which ectopically express Cdk4 and hTERT) were from the Hamon Center cell line repository (UT Southwestern Medical Center; Gazdar et al., 2010 (link); Ramirez et al., 2004 (link)). All cell lines were DNA fingerprinted for provenance (PowerPlex 1.2 Kit; Promega) and were mycoplasma free (e-Myco Kit; Boca Scientific). Fluorescent BODIPY-FA was from Molecular Probes, pBabePuro and pBabePuro-KRAS-G12V were from Addgene, the ACSL3 cDNA was from Dr. Takahiro Fujino (Department of Bioscience, Ehime University), and pGL3-ACSL3luc was from Dr. Jingwen Liu (Department of Veterans Affairs Palo Alto Health Care System). We performed site-directed mutagenesis using the Quickchange kit (Agilent). See also Supplemental Information for tissue culture and assay conditions.

The HCC1833 neuroendocrine NSCLC (NE-NSCLC) line was obtained from the Hamon Cancer Center Collection (University of Texas Southwestern Medical Center), maintained in RPMI-1640 medium supplemented with 5% FBS without antibiotics at 37 °C in a humid atmosphere containing 5% CO₂ and 95% air. Cells lines were DNA-fingerprinted using Powerplex 1.2 kit (Promega), and mycoplasma was tested using an e-Myco kit (Boca Scientific). Cells lines were grown up to 70% confluence before splitting. ABT-263, a potent small molecule inhibitor of Bcl-2 family, was initially dissolved in DMSO and warmed at 37 °C for 10 min in an ultrasonic water bath. The stock solution was then diluted serially to different concentrations till a final concentration of 1 μM. The drug with final concentration of 1 μM was introduced to the cell culture after the cells were adhered to the culture plate (~4.5 h after plating). Each culture plate (for varying time points) was maintained at 2 to 3 million (10⁶) cells/mL cell density and was treated 1 μM of ABT-263, clinically relevant concentration that is probably saturating for Bcl-2 family binding.

HBEC30KT and the NSCLC cancer cell lines were generated as previously described (20 (link)). HBEC3KT and their oncogene-transformed derivatives were developed by the Minna lab (22 (link)). All NSCLC lines used in this study were obtained from the Hamon Cancer Center Collection (UT Southwestern Medical Center) and maintained in RPMI-1640 (Life Technologies) supplemented with 5% FCS at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. All cell lines have been DNA fingerprinted using the PowerPlex 1.2 Kit (Promega) and are mycoplasma free using the e-Myco Kit (Boca Scientific). Culture media were purchased from Life Technologies. Human bronchial epithelial cell (HBEC), NSCLC, and Human retinal epithelia ARPE-19 cell lines were obtained from the ATCC and cultivated in complete KSF medium, RPMI-5% FBS (Sigma), or in DMEM/F12-10% FBS, respectively.

Human pancreatic cancer cell lines (AsPC-1, Panc-1, Capan-1, and Mia PaCa-2) were obtained from ATCC (Manassas, Virginia), and a murine cell line Pan02 was obtained from the Developmental Therapeutics Program at the National Cancer Institute at Frederick, Maryland. KPC-M09 cells were derived from KPC PDA mice as described in the supplementary methods. All cell lines were cultured in DMEM (Invitrogen) or RPMI (Invitrogen) containing 10% FBS and maintained in a humidified atmosphere with 5% CO₂ at 37°C. The human cell lines were DNA fingerprinted for provenance using the Power-Plex 1.2 kit (Promega) and confirmed to be the same as the DNA fingerprint library maintained by ATCC. All cell lines were confirmed to be free of mycoplasma (e-Myco kit, Boca Scientific) before use.

Immortalized normal human bronchial epithelia cells (HBECs) with stable knockdown of TP53 with shRNA (HBEC-shp53) were provided by John Minna at UTSW (21 (link)–23 (link)). Ectopic expression of KRAS^G12V in HBEC-shp53 was performed with lentiviral infection followed by selection with hygromycin B for 7 days. HBEC-shp53-PCDH7 or HBEC-shp53-KRAS^G12V-PCDH7 cells were established with the pLX303-PCDH7 lentivirus followed by selection with blasticidin for 10 days. All HBECs were cultured in keratinocyte serum-free medium (KSFM; Life Technologies Inc.) containing 50 μg/mL of bovine pituitary extract (BPE; Life Technologies, Inc.) and 5 ng/mL of EGF (Life Technologies, Inc.). Human lung adenocarcinoma H1944 cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 100 units/ml of penicillin and streptomycin (Life Technologies, Inc.). PCDH7 depletion in H1944 cells was achieved using Lenti-CRISPRv2 followed by selection with puromycin for 7 days (1 μg/mL). All cell lines used in this study were cultured in 5% CO2 at 37C, tested negative for mycoplasma contamination, and were authenticated in 2016 with the PowerPlex 1.2 kit (Promega).