Methotrexate Is a JAK/STAT Pathway Inhibitor
Fig 3
The effect of methotrexate on human HEL cells homozygous for the activating JAK2 V617F mutation.
A) HEL cells treated with the indicated concentrations of methotrexate and blotted to show levels of total STAT5 (tSTAT5), phosphorylated STAT5 (pSTAT5), total STAT3 (tSTAT3), phosphorylated STAT3 (pSTAT3) and β-actin shown as a loading control. Levels of pSTAT5 and pSTAT3 are clearly reduced at most drug concentrations. B & C) Quantification of pSTAT5 (B) and pSTAT3 (C) levels present in HEL cells treated with the indicated concentrations of methotrexate. D) HEL cells treated with the indicated concentrations of methotrexate and blotted to show levels of total STAT5 (tSTAT5), phosphorylated STAT5 (pSTAT5) and β-actin shown as a loading control. Indicated lanes have been grown in the presence of 0.3μg/ml folinic acid. Suppression of pSTAT5 levels is seen at most drug concentrations. E) HEL cells treated with the indicated concentrations of methotrexate and blotted to show levels of total STAT5 (tSTAT5), phosphorylated STAT5 (pSTAT5) and β-actin shown as a loading control. Right hand lanes have also been treated with 15ng/ml recombinant human erythropoietin (EPO). Blots have been underexposed to show the difference in pSTAT5 levels and so are not comparable to other panels. F) HEL cells treated with the indicated concentrations of methotrexate or ruxolitinib and blotted to show levels of total STAT5 (tSTAT5), phosphorylated STAT5 (pSTAT5) and β-actin shown as a loading control. Levels of pSTAT5 are reduced at most drug concentrations. In the representative western blots shown the position and apparent molecular weights of markers used are indicated in kDa. For graphs levels have been normalised to carrier control (0μM drug) and error bars show the standard deviation of three independent experiments. *** = p < 0.001, ** = p < 0.01, * = p < 0.05.