Page 142 - 2020_01-Haematologica-web

A. Tobio et al.
AB
C
D
EF
Figure 6. MEK/ERK signaling pathway co-operates with JAK2 in the regulation of STAT5 to induce IL-6 while PI3K regulates IL-6 independently of STAT5. (A) Effect of inhibitors for KIT (KIT-I: dasatinib; 0.5 mM), JAK2 (JAK2-I: fedratinib; 1 mM), PI3K (PI3K-I: LY294002; 10 mM), MEK/ERK1/2 (ERK-I: U0126; 10 mM), STAT5 (STAT5- I: CAS285986-31-4; 50 mM), or their combination on the protein and phosphorylation levels of STAT5 in HMC-1.2 cells. (B) Effect of inhibitors of JAK2 and ERK1/2 alone or in combination on STAT5 phosphorylation in HMC-1.2 cells. (A and B) Lysates were obtained after 2 hours (h) incubation with the indicated inhibitors in serum-free medium. Data under the blots are the mean±Standard Error of Mean (SEM) of three independent experiments carried out in duplicates, and represent fold change in the relative band fluorescence compared to the untreated. STAT5 content was used to normalize the data. (C) Inhibition of MEK/ERK1/2 reduces STAT5A and STAT5B expression ((left and right, respectively) after 16 h incubation. Relative expression of STAT5 mRNA was obtained by comparison with the expres- sion of GAPDH using the DCt method and the results are expressed as fold change compared to untreated cells at each time (2 h or 16 h). (D and E) Inhibitors for STAT5, PI3K and MEK/ERK1/2 additively prevent IL-6 mRNA induction (D, left) and IL-6 release in HMC-1.2 cells (D, right) and P815 cells (E). The release of IL-6 into the media was determined after 6 h. (F) Inhibitors for JAK2, PI3K and MEK/ERK1/2 additively prevent IL-6 mRNA induction in HMC-1.2 cells. All data represent mean±Standard Error of Mean of at least three independent experiments. *P=0.01, **P<0.01, ***P<0.001 and ****P<0.0001.
132
haematologica | 2020; 105(1)