Fig 1: Characterization of the pemetrexed-resistant A549 and CL1 lung cancer cells. The expression of TS, DHFR, FPGS and GGH were detected by western blot assay in A549 (a) and CL1 (b) sublines. Inhibition of TS by shRNA in CL1/A100 (c) and CL1/A200 (d) sublines were followed by MTT assay. The sensitivities of TS-inhibited CL1 sublines to MTX treatment were examined by MTX (e) and 5-FU (f). *P<0.05 was considered statistically significant. Individual clones are identified by their unique TRC number, which were purchased from National RNAi Core Facility at Academic Sinica, Taiwan. To inhibit TS expression, the CL1 sublines (5 × 104 per well) were sub-cultured onto 60-mm plates. After 16 h of incubation at 37 °C, cells were infected with recombinant lentivirus vectors with protamine sulfate added (0.8 µg/µl) to help infection. The next day, the medium was removed and the cells were selected by 2 µg/ml puromycin (Sigma, P8833) at 37 °C for 48 h, followed by MTT assay or protein detection. The clone of shGFP (TRCN0000072178) for vector control targeted to GFP, whereas the clones of shTS-1 (TRCN0000291720), shTS-2 (TRCN0000291655) and shTS-3 (TRCN0000045665) targeted to TS.