Tet free fetal bovine serum

The sublines U251tetEHD3 and U87tetEHD3 cell lines derived from the glioma cell lines U251 and U87MG respectively to express EHD3 upon Doxycycline (Dox) induction were previously described [8 (link)]. The cells were authenticated by the WSU Applied Genomics Technology Center (AGTC)(http://agtc.wayne.edu/cell-lines.php) by short tandem repeat profiling using the Promega GenePrint 10 kit. These cells were maintained in DMEM with l-glutamine (Life Technologies, Inc., Invitrogen Corp.), 10% Tet-free fetal bovine serum (Clontech) and 1% penicillin-streptomycin (Life Technologies, Invitrogen) and grown at 37°C. Induction of EHD3 expression is obtained by treating cells with 2 mg/ml of Dox for the indicated time. U251MG cells conditionally expressing the EGFRvIII mutant using the T-Rex Tet-on system (Invitrogen) were a kind gift from Dr Amyn Habib (University of Texas Southwestern Medical Center) and were described previously [59 (link)].

The stable cell lines were constructed by transduction of U937 cells with a retroviral vector system and by selection of single clones. In brief, the N-terminal HA-tagged AML1-ETO and ETO2-GLIS2 cDNAs were subcloned into a pRetrox-Tet3G vector that was equipped with puromycin resistance. The retroviruses were produced by co-transfection of 293T cells with the pRetrox-Tet3G and the psPAX2 and pMD2.G helper vectors. U937 cells were infected with filtered virus-containing supernatant of the 293T cultures, in the presence of 8 μg/ml polybrene (Sigma, H9268), and centrifuged at 1200 × g, 37°C for 90 min. Infected cells were selected with 1 μM puromycin at 12–24 h after infection. The survived cells were then subjected to a limiting dilution method in 96-well plates to obtain single cell clones of stable cell lines. To avoid the leaky expression before induction, the stable cell lines were grown in RPMI 1640 medium (ThermoFisher Scientific, C11875500CP) supplemented with 10% Tet-free fetal bovine serum (Clontech, 631106).