Figure 3.

mTOR inhibitor PP242 up-regulated EGFR expression and phosphorylation but had no significant effect on the downstream signaling pathways of EGFR in HT-29 cells. HT-29 cells were untreated or treated with 20 µg/mL CTX and 1 µmol/L PP242 alone or in combination for 96 h. (A) The protein content of cellular total and phosphorylated EGFR, MEK, ERK, MKK and JNK contents were determined by western blotting. (B) The semi-quantitative analysis of cellular total and phosphorylated EGFR, MEK, ERK, MKK and JNK contents was performed based on the relative density values of the bands of western blotting. The relative density value of the control group was set to one. (C) qPCR was used to measure the mRNA expression of EGFR, MEK, ERK, MKK and JNK in different treatment groups relative to the control group. The relative mRNA expression of the control group was set to one. Each bar represents the mean ± SEM. All data were derived from at least three separate experiments. *, P<0.05; **, P<0.01; ***, P<0.001. mTOR, mammalian target of the rapamycin; EGFR, epidermal growth factor receptor; CTX, cetuximab; MEK, mitogen-activated protein kinase kinase; ERK, extracellular regulated protein kinase; MKK, MEK 4/7; JNK, c-Jun N-terminal kinase; qPCR, quantitative real time polymerase chain reaction; SEM, standard error of mean.