Page 140 - 2020_01-Haematologica-web

A. Tobio et al.
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Figure 4. MEK/ERK-, PI3K- and JAK2-mediated pathways are independently activated by D816V-KIT and contribute to ligand-independent IL-6 induction. (A and B) Effect of inhibition of MEK/ERK1/2 (U0126), p38 (SB203580) (A) or PI3K pathways (LY294002) (B) on the expression of IL-6 mRNA (left) after treatment for 2 hours (h) and the release of IL-6 into the media (right) by HMC-1.2 cells for 16 h. (C) Effect of the JAK2 inhibitor fedratinib at the indicated concentrations on the expression of IL-6 mRNA (left) and the release of IL-6 into the media (right) by HMC-1.2 cells. (D) Effect of various concentrations of inhibitors for JAK1/2 (ruxolitinib) and JAK3 (tofacitinib) on the expression of IL-6 mRNA by HMC-1.2 cells. (E) Effect of inhibition of MEK/ERK1/2, PI3K or JAK2 on the production of IL-6 by mouse P815 cells after 6 h of incubation. (F) Effect of inhibitors for KIT (KIT-I: dasatinib; 0.5 mM), JAK2 (JAK2-I: fedratinib; 1 mM), PI3K (PI3K-I: LY294002; 10 mM) and MEK/ERK1/2 (ERK-I: U0126; 10 mM) on the phosphorylation of KIT, JAK2, AKT and ERK1/2 in the indicated amino acid residues. Inhibitors were incubated for 2 h in serum-free medium. Numbers under each phosphorylated band are Standard Error of Mean (SEM) of at least three experiments and represent fold changes in the relative band fluorescence (normalized to the corresponding total expression) compared to untreated cells. Relative expression of IL-6 mRNA was obtained by comparing to the expression of GAPDH using the DCt method and the results were expressed as fold change compared to untreated cells. Data represent the mean±SEM and are from three independent experiments. **P<0.01, ***P<0.001 and ****P<0.0001.
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haematologica | 2020; 105(1)