DHFR was identified as the top candidate during a large-scale screen against methotrexate. (a) Immunoblotting showing the conditional expression of DHFR in HEK293_M2 cells. Addition of 4 ng/ml, 16 ng/ml, 63 ng/ml and 2,000 ng/ml of doxycycline (dox) induced DHFR protein expression in the stable cell line HEK293_M2. Alpha tubulin was used as a loading control. For clarity, protein expression level at 0.0032 ng/ml of doxycycline has been omitted. (b) Single DHFR overexpression rescued methotrexate toxicity in HEK293_M2 cells. The gene coding for DHFR was cloned into the lentiviral vector and used to create stable HEK293_M2 cells able to conditionally express DHFR. Dose-response curves of the cells grown in the presence of an increasing concentration of doxycycline were calculated after 2 days of exposure to 15, 30, 60, 125, 250, 500 and 1,000 nM of methotrexate and compared with those for cells cultured in the presence of DMSO as control. Cell survival was quantified using a SRB assay. Errors bars represent the standard deviation for triplicate assays. (c) Identification of genes conferring resistance to methotrexate when overexpressed. HEK293_M2 cells harboring the 12,212 hORF collection were grown in the presence of a lethal dose of methotrexate. The nature of the hORFs conferring resistance to the drug was identified by plotting the log2 of the signal intensity for each hORF in the cells cultured in the presence of methotrexate on the x-axis; and by plotting the log2 ratio of the signal intensity for each hORF of the cells cultured in the presence of the drug divided by the signal intensity for each hORF of the cells grown in the presence of DMSO on the y-axis.