Figure 1.

Methotrexate is a JAK/STAT inhibitor that reduces hJAK2 V617F-induced erythrocytosis in vivo. (A,B) Western blots of the indicated total (t) and phosphorylated (p) STAT proteins in extracts from HEL cells (A) or the spleens of 10–11 week old mice of the indicated genotypes (B) following treatment with the indicated concentrations of MTX and rux. β-ACTIN serves as a loading control and apparent molecular weights are indicated in kDa. (C) Levels of PIM1 messenger ribonucleic acid (mRNA) expressed by spleen cells harvested from mice of the indicated genotype and drug treatments. Results are expressed as a fold change following normalization to β-actin mRNA and PBS-treated wild-type mice. (D) Hemoglobin concentration in blood from individual 10–11 week old mice of the indicated genotypes treated with either phosphate-buffered saline carrier control (PBS, gray), methotrexate (MTX, green) or ruxolitinib (rux, orange) for 28 days. Individual values, mean and standard deviations are shown. Samples were compared by one-way ANOVA. (E) Hematoxylin and eosin stained sections through decalcified tibia from mice of the indicated genotypes treated with the indicated compounds for 28 days. No marrow fibrosis was observed in either treated or untreated homozygous animals. Scale bar is 50μm. Images were obtained from a Zeiss Axioskop 2 with a 20×/0.5NA objective, a MicroPublisher 5.0 RTV camera and Qimaging v3.1.3.5 software. Brightness and contrast were adjusted in Photoshop CS5.