Fig 1.
Identification of methotrexate and aminopterin as suppressors of the Drosophila JAK/STAT pathway.
A) The chemical structures of methotrexate (top) and aminopterin (bottom). B) A rank plot of the compounds screened using the 6x2xDrafLuc reporter system. Compounds are ranked according to the z-scores of their interactions with a score >3 indicating a significant increase in activity and an score <-3 indicating a significant suppression. The position of aminopterin and methotrexate are indicated. C-D) Normalised 6x2xDrafLuc transcriptional reporter activity in Drosophila Kc167 cells treated with the indicated concentrations of aminopterin and stimulated with the pathway ligand Unpaired (Upd) (C) or by transfection with the gain-of-function JAK mutant HopTuml (D). E-F) Normalised 6x2xDrafLuc transcriptional reporter activity in Drosophila Kc167 cells treated with the indicated concentrations of methotrexate and stimulated with Upd or HopTuml. In graphs reporter activity has been normalised to carrier control (0 μM drug) and error bars show the standard deviation of four independent experiments. *** = p < 0.001, ** = p < 0.01, * = p < 0.05.
Fig 2.
The effect of methotrexate on the constitutively active JAK/STAT pathway present in human HDLM-2 cells.
A) HDLM-2 cells treated with the indicated concentrations of methotrexate and aminopterin and blotted to show levels of total JAK1 (tJAK1), dual phosphorylated JAK1 (ppJAK1), total JAK2 (tJAK2), dual phosphorylated JAK2 (ppJAK2) and ß-actin shown as a loading control. Levels of ppJAK1 and ppJAK2 are reduced at high drug concentrations. B-C) Quantification of ppJAK1 (B) and ppJAK2 (C) following treatment with methotrexate and aminopterin. D) HDLM-2 cells treated with the indicated concentrations of methotrexate and aminopterin and blotted to show levels of total STAT1 (tSTAT1), phosphorylated STAT1 (pSTAT1), total STAT3 (tSTAT3), phosphorylated STAT3 (pSTAT3), total STAT5 (tSTAT5), phosphorylated STAT5 (pSTAT5) and β-actin shown as a loading control. Levels of pSTAT1 and pSTAT5 are reduced at most drug concentrations while levels of pSTAT3 appear to be unaffected. E-G) Quantification of pSTAT1 (E), pSTAT3 (F) and pSTAT5 (G) following treatment with methotrexate and aminopterin. H) Human HDLM-2 cells treated with the indicated concentrations of methotrexate and blotted to show levels of phosphorylated AKT (pAKT), phosphorylated c-Jun (p c-Jun), phosphorylated ERK1/2 (pERK1/2) and β-actin shown as a loading control. The levels of each phospho-protein tested appears to be unaffected by methotrexate. 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.
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.