PV and ET labeled protein pools and the internal standard protein samples, were

treated cultures, However, the same differences were not seen when treated ET samples were compared with untreated cells, Figure 4A B shows the flow cytometry results for the CD34 BFU E cultures. CBA analysis showed an important decrease of phospho STAT1 in PV samples patients, however, we found no sig nificant differences in phospho STAT1 with and with out KNK437 treatment in ET patients. We define ratios as concentration ratios of phosphoproteins normalized with non phosphoproteins as total protein numeric value. Add itionally, phospho MEK showed under expression after KNK437 treatment, and this was more pronounced in sam ples from PV patients vs. ET patients, Moreover, the other MAPK phospho protein, phospho p38, was differentially expressed with and without KNK437 treatment in samples from PV patients, but was unchanged in ET patients. Phospho AKT showed no decrease with treat ment. A full list of the proteins and phospho proteins ex pression sample per sample is summarized in Additional file 5: Table S5. HSP70 inhibition in an ex vivo cell line To confirm the molecular mechanism of the HSP70 in hibitor in the JAK2 STAT and MAPK pathways, we per formed Western blot on HEL and Ba F3 JAK2 V617F cell lines proteomes, with and without KNK437 treatment, This showed a reduction of the phospho JAK2 and phospho STAT5 protein with treatment, but no reduction of phospho ERK and phospho p38. ImageJ quantification confirmed these results and showed a 50% reduction in the expression of phospho JAK2 in the HEL cell line fol lowing treatment with KNK437. Additionally, HSP70, HSP90 and detection by Western Blot showed a slight decrease of HSP70 expression after KNK437 treatment, but no significant difference in HSP90 expression in the HEL cell line, with and without KNK437 treatment, KNK437 decreased the activation of JAK2 as well as its ex pression. This decrease in JAK2 expression resulted in the inhibition of leading proliferative pathways related to JAK2 GATA1 also showed no differential ex pression with the HSP70 inhibitor treatment, Similarly to the primary BFU E, incuba tion with the HSP70 inhibitor KNK437 in HEL and Ba F3 JAK2 V617F caused a reduction of 20 50% in the cell viability, In order to validate the KNK437 inhibition on HSP70, and check the specificity of this treatment, additional HSP70 interference was performed with specific a siRNA, The results showed a proper interference, de creasing the protein levels of HSP70, but not HSP90. Be sides, HSP70 interference assay produces the decrease of the expression of JAK2, and the inhibition of JAK STAT signaling due to the decrease of phospho STAT5. Discussion Many authors believe in the possibility of other events and or genetic alterations upstream of the JAK2 muta tion in MPN, This opens new frontiers in the pathogenesis of the disease and the phenotypic diver gence among the different MPNs must be studied to find new defective molecules that may potentially be used for novel targeted therapies. Proteomic screening to find new molecular targets has been an under used strategy in MNP. This may be due to several factors, namely the difficultly in selecting the correct target cell populations and their protein fractions, or the lack of a high quality protein extraction technique.