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. 2014 Dec 31;6(2):1128–1142. doi: 10.18632/oncotarget.2731

Figure 5. Effects of Ras/ERK1/2 inhibition on STAT3 phosphorylation and IDO expression in mesothelioma cells.

Figure 5

HMM samples (UPN: unknown patients number), treated with a TetON vector containing a shRNA for RAS (sh) or with the ERK1/2 inhibitor PD98059 (10 μmol/L for 24 h, PD), were cultured in medium without (−) or with (+) 1 μg/mL doxycycline (doxy). After 24 h, the following investigations were performed. (a) The expression of Ras, phospho(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2, total ERK1/2 was measured in whole cell lysates by Western blotting. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (b) The ERK activity was measured in cell lysates using a specific ELISA kit. Data are presented as means ± SD (n = 3). Vs untreated (doxy -, PD -) cells: *p < 0.001. (c) The expression of phospho(Tyr705)-STAT3, phospho(Ser727)-STAT3, total STAT3 was assessed in whole cell lysates by Western blotting. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (d) The relative expression levels of IDO mRNA were measured by qRT-PCR (open bars, mRNA); the kynurenine level in cell culture supernatant, taken as an index of IDO enzymatic activity, was measured fluorimetrically (solid bars, act). Data are presented as means ± SD (n = 4). Vs untreated (doxy -, PD -) cells: *p < 0.005.