Figure 2. p-p38 MAPK and p-EGFR are up-regulated in PTX resistant cell lines.

A. The activities of signaling molecules related to cancer cell survival and the development of drug resistance were investigated in parental cells and PTX resistant cells. Activities were estimated by calculating the ratio between phosphorylation and total protein levels based on the band intensity of at least three immunoblots using ImageJ software (National Institutes of Health freeware). The right panel exhibits the representative immunoblots of H226B and H226B/R cells. The data are presented as the mean ± SD. The statistical significance was analyzed using Student's t-test (NS = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001 vs. the respective control). B. H460/R and H226B/R cells were incubated with erlotinib (ETN) at the indicated concentrations for 24 h. The expressions of p-p38, p-EGFR and their total proteins were analyzed using western blot analysis. (C and D) H460/R and H226B/R cells were treated with SB203580 (SB; 50 μM) at the indicated time points. C. The expression of p-EGFR, p-p38 and their total proteins were analyzed using western blot analysis. D. The mRNA level of EGFR was determined using RT-PCR. E. The basal expression levels of EGFR mRNA between parental cells and PTX resistant cells were detected using RT-PCR (lower) and confirmed by quantitative RT-PCR (upper). The data are presented as the mean ± SD. The statistical significance was analyzed using Student's t-test (*** P < 0.001 vs. the respective control). F. H460/R and H226B/R cells were stably transfected with a dominant-negative mutant of p38 MAPK (p38 DN). Western blot analysis was performed to investigate the expression changes of p-EGFR and EGFR following the transfection of inactive p38 MAPK (upper). RT-PCR was also conducted to verify the influence of p38 MAPK on the expression of EGFR mRNA (lower).