Cell id system

Human peritoneal mesothelial LP9/TERT-1 (LP9) cells [13 (link)] were purchased from Brigham and Women’s Hospital, Harvard University, Boston, MA and grown as reported previously. Human MM cell lines, H2373, H2595, H2461, H2818, H2596 and HP-1 were contributed by Dr. Harvey Pass (New York University, New York, NY) [14 (link)]. Hmeso cells were isolated by Reale et al. [15 (link)]. Immortalized mesothelial cells (LP9) were similar to primary mesothelial cells in their responses to asbestos as previously published [8 (link)]. All cells were cultured as reported previously [16 (link)]. Cell lines were validated by STR DNA fingerprinting using the Promega CELL ID System (Promega, Madison, WI) as previously reported [17 (link)]. Human MM tumor tissues and normal counterparts were also obtained from Dr. Pass [18 (link)], without revealing their identity. Doxorubicin (Dox) was purchased from Sigma (St. Louis, MO) and cisplatin from Alfa Aesar (Ward Hill, MA). Dox and cisplatin concentrations for the present study were selected based on already published literature for MM [16 (link), 19 (link)] or other cell lines [20 (link)].

The human cervical carcinoma cell lines, HeLa (ATCC CCL-2) and CaSki (ATCC CRL-1550), and normal primary (ARPE19) (ATCC CRL-2302) and immortalised (hTERT-RPE-1) (ATCC CRL-4000) epithelial cells were obtained from the American Type Culture Collection (ATCC). Cancer cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml) and 10% Fetal Calf Serum (FCS) (HiClone, Thermo Scientific, USA). Normal cells were cultured in DMEM/F12 media, supplemented with penicillin, streptomycin and 10% FCS. hTERT-RPE-1 cells were maintained in the presence of 10 μg/ml Hygromycin B. Cells were incubated at 37 °C in 5% CO₂. Cancer cell lines were authenticated by DNA profiling using the Cell ID system (Promega, USA).

PC9 was kindly provided by Dr. Yukito Ichinose (National Hospital Organization Kyushu Cancer Center, Fukuoka, Japan) [49 (link)]. HCC827 was purchased from the American Type Culture Collection. PC9 and HCC827 were not further tested or authenticated by the authors. These lung cancer cell lines were maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS) and incubated in a humidified atmosphere containing 5% CO2 at 37°C. Afatinib-resistant sublines were established from HCC827 cells as previously described [8 (link), 29 (link), 30 (link)]. We cultured HCC827 cells in increasing, step-wise concentrations of afatinib up to 1 μmol/L over the following 11 months. We independently cloned two afatinib-resistant sublines from two dishes and designated them HCC827/BR1-8 and HCC827/BR2-3. The identities of these sublines were confirmed by analyzing their short tandem repeat profiles using the Cell ID System (Promega, Madison, WI). All cell lines were passaged for ≤6 months.

MDA-MB-231 (basal, ER-, PR-, HER2-), MDA-MB-453 (luminal, ER-, PR-, HER2-low), MDA-MB-468 (basal, ER-, PR-, HER2-), MCF-7 (luminal, ER+, PR+, HER2-), BT-474 (luminal, ER-, PR-, HER2+) and SK-BR-3 (luminal, ER-, PR-, HER2+) mammary carcinoma cell lines were obtained from ATCC (LGC Standards) and grown in Dulbecco modified Eagle medium (DMEM, Lonza) supplemented with 10% fetal bovine serum (FBS, SIGMA).
STAT3-silenced mammary carcinoma cells were generated by lentiviral transduction of pLKO vectors encoding for human shRNAs of the MISSiON system (sh2_TRCN0000020842, sh3_TRCN0000020843, SIGMA).
All cell lines were authenticated by BMR Genomics srl Padova, Italia, on January 2012 according to Cell ID ^™ System (Promega) protocol and using Genemapper ID Ver 3.2.1, to identify DNA STR profiles.

To isolate afatinib-resistant cell lines, we cultured in increasing, step-wise doses of afatinib up to 1 μM over the following 11 months, and PC9 BR(3Mo), PC9BR(10Mo), and PC9BR(11Mo) were established (13 (link), 19 (link)). We also established the revertant cells, PC9BR (21Mo), by culturing PC9BR (11Mo) cells under drug-free condition for 10 months and generated the subclones Rev1 from PC9BR (21Mo). Using limiting dilution, we further generated the clones B3, B19 and B20 from PC9 BR (11Mo). The identity of these clones was confirmed by analyzing their short tandem repeat profile using the Cell ID System (Promega, Madison, WI).