Figure 4

DPD expression modulated cellular sensitivity to gemcitabine. (a) (left panel) UM-UC-3 cells were transfected with 25 nM each of the indicated siRNA and incubated for 72 h. Whole-cell extracts from UM-UC-3 cells were subjected to SDS-PAGE followed by western blotting for the indicated proteins. (right panel) UM-UC-3 cells transfected with siRNA were incubated with 0 or 22.0 μM gemcitabine. After 48 h, cell numbers were counted. Relative cell counts when transfected with control siRNA were defined as 1. Boxes, mean; bars, ± SD. **P < 0.01 (one-way ANOVA). (b) (left panel) Whole-cell extracts from HEK293 cells and DPD-overexpressing HEK293 cells were subjected to SDS-PAGE followed by western blotting for the indicated proteins. (right panel) HEK293 cells and DPD-overexpressing HEK293 cells were incubated with 0 or 6.67 nM gemcitabine. After 48 h, cell numbers were counted. Relative cell counts of HEK293 cells were defined as 1. Boxes, mean; bars, ± SD. ***P < 0.001 (Student’s t-test). (c) (left panel) HEK293 cells were transfected with 5 μg each of the indicated plasmid and incubated for 72 h. Whole-cell extracts from HEK293 cells were subjected to SDS-PAGE followed by western blotting for the indicated proteins. (right panel) HEK293 cells were transfected with 5 μg each of the indicated plasmid and incubated with 0 or 33.3 nM gemcitabine. After 48 h, cell proliferation was measured with CellTiter Glo. Relative cell proliferation when transfected with wild-type DPYD expression plasmid was defined as 1. Boxes, mean; bars, ± SD. *P < 0.05 (Student’s t-test.). (d) (left panel) After extraction of total RNA from parental (RT112-P and UM-UC-13-P) and gemcitabine-resistant RT112 (RT112 GEM-R) and UM-UC-13 (UM-UC-13 GEM-R) cells and DNA synthesis, quantitative real-time PCR was performed for DPYD and 18 s ribosomal RNA. Each target transcript level was corrected relative to the corresponding 18 s ribosomal RNA transcript level. The level of each target transcript in parental cells was defined as 1. Boxes, mean; bars, ± SD. ***P < 0.001 (Student’s t-test). (right panel) Whole-cell extracts from parental and GEM-R RT112 and UM-UC-13 cells were subjected to SDS-PAGE followed by western blotting for the indicated proteins. (e) (left panel) RT112 GEM-R cells were transfected with 25 nM each of the indicated siRNA and incubated for 72 h. Whole-cell extracts from RT112 GEM-R cells were subjected to SDS-PAGE followed by western blotting for the indicated proteins. (right panel) RT112 GEM-R cells were transfected with siRNA and incubated with 0 or 10.0 μM gemcitabine. After 48 h, cell numbers were counted. Relative cell counts when transfected with control siRNA were defined as 1. Boxes, mean; bars, ± SD. *P < 0.05, **P < 0.01 (one-way ANOVA).