Figure 4.

Gemcitabine induces ERK phosphorylation and MICA/B expression by activating TAK1. (a) HeLa cells were treated with the indicated concentrations of gemcitabine for 24 hr or treated with gemcitabine (2 μM) for the indicated lengths of time. Cell lysates were prepared and analyzed for TAK1, ERK, JNK, p38, and p65 phosphorylation as well as their total proteins by Western blot. (b) HeLa cells were incubated for 24 hr in the absence or presence of gemcitabine (2 μM) minus or plus 5Z (5 μM). Alternatively, HeLa cells were transfected with a scrambled control siRNA or TAK1 siRNA. After incubation for 24 hr, the cells were left untreated or treated with gemcitabine (2 μM) for another 24 hr. Cell lysates were prepared and analyzed for TAK1, ERK, JNK, p38, and p65 phosphorylation and their total proteins by Western blot. Relative phosphorylation levels were analyzed by quantifying the density of phosphorylated protein bands normalized by the density of their corresponding total protein bands with NIH Image-J software and presented as bar graphs. Data are the mean ± SD of three experiments. **p < .01, compared to the untreated control. ^##p < .01, compared to the corresponding no-inhibitor or control siRNA-transfected controls. (c & d) HeLa cells were treated with gemcitabine minus or plus 5Z (c) or transfected with TAK1 siRNA and treated with or without gemcitabine as described above (d). Single-cell suspensions were prepared and analyzed for MICA/B expression by flow cytometry. Green line, isotype control; Red line, MICA/B. The data represent one of three independent experiments with similar results.