FIGURE 5.

The effect of a bicyclic peptide (16) on ERK and p38 pathways. (A) Serum-starved HEKT cells were pre-treated with 0, 25, 50, 100 µM peptide 16 for 2 h, and then stimulated by 50 ng/ml EGF for 15 min or 200 ng/ml EGF for 10 min. Samples were subjected to Western-blot analysis; activity of the ERK pathway was followed by anti-pp-ERK and anti-ERK antibody; anti-tubulin antibody was used as load control and demonstrates equal sample load in addition to the anti-ERK Western-blot signal. Bar charts show the ppERK2/ERK2 intensity ratio of each sample. (B) Serum-starved HEKT cells were pre-treated with the peptide 16 at 0.1, 1, 3, 10, 25, 100 µM for 1 h and then stimulated by 0.4 M Sorbitol for 10 min (upper panel) or treated only with the medium (lower panel; Mock). p38 activation was confirmed by using anti-pp-p38 antibody, and the total p38 amount of the samples by using the anti-p38 antibody. The graph shows the pp-p38/p38 intensity ratio of each band obtained from the densitometry of Western-blots in the case of Sorbitol (in grey) or medium (Mock; in black) treatment (C) Cell viability test with peptide 16. HEKT cells were incubated with the peptide at 10 and 100 µM for 24 h, then the viability of cells was determined by PrestoBlue™ reagent according to the manufacturer’s instructions (ThermoFisher Scientific, #A13261). Note that peptide incubation times before stimulation was only up to 2 h at maximun on Panel A and B. Error bars show SD of 3 independent measurements. *Indicates statistical significance between peptide-treated and control (p < 0.05 with two-sided independent Student’s t-test).