Methotrexate Is a JAK/STAT Pathway Inhibitor
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.