Figure 1. ChlR1-depleted cells are sensitive to cisplatin.

(A) HeLa cells were infected with lentivirus expressing the indicated shRNAs and selected in the presence of puromycin for stable expression of shRNAs. Western blotting results show that Timeless or ChlR1 was efficiently downregulated. (B, C) Cells infected with the indicated shRNA-expressing lentivirus were treated with vehicle (water in B) or 100 μM cisplatin (in C) for one hour and returned to fresh medium. Cells were counted at the indicated time in triplicate, and percentages of cell growth were calculated against the number of cells at the 0 h time point. The error bars represent standard deviation. (D) HeLa cells stably expressing shRNAs used above were incubated with the indicated concentrations of cisplatin for 1 hour. Cells were then washed and released into fresh medium for clonogenic survival assays as described in Material and Methods. (E) HeLa cells were transiently transfected with a plasmid vector expressing ChlR1 shRNA (ChlR1-5) and incubated with the indicated concentrations of cisplatin for 24 hours. Cells were washed and returned to growth medium for clonogenic survival assay as described in D. The error bars represent standard deviation obtained from three independent experiments. Western blotting results show efficient ChlR1 depletion by the vector expressing ChlR1 shRNA.