Figure 5.

The synergistic effects on the inhibition of cell proliferation by RP4010 and gemcitabine/nab-paclitaxel are mediated through the downregulation of mTOR and NFAT/NF-κB signaling. (A) MiaPaCa-2 cells were treated with either 10 µM RP4010 alone or in combination with 300 nM gemcitabine and 3 nM nab-paclitaxel. After 48 h, RNA was isolated and RT-qPCR was performed as described in Methods (** p < 0.01). (B) MiaPaCa-2 cells were grown overnight in 100-mm petri dishes to around 50% confluence. On the next day, the cells were then incubated with 10 µM RP4010 for 72 h. Extraction of total protein and determination of its concentration were carried out as described in the Methods. The protein lysates were subjected to Western blotting using antibodies against NFAT1, NF-kB, and p-S6K. GAPDH was used as a loading control (n = 1). (C) Expression of each protein was indicated as fold change relative to the control and a quantitative analysis of mean pixel density of the blots was performed using ImageJ software.