Page 115 - 2020_08-Haematologica-web

PDGFRb regulation in murine myelofibrosis
ential expression of all classical PTP and the dual-specific phosphatase Pten. These analyses included the six PTP which are known to target PDGFRb.19-22 However, Ptpn1 (encoding PTP1B), Ptpn2 (encoding TC-PTP), Ptpn6 (encoding SHP-1), Ptpn11 (encoding SHP-2), Ptpn12 (encoding PTP-PEST), and Ptprj (encoding DEP-1) were not differentially expressed in the bone marrow of early fibrotic, 10-month-old mice (data not shown). To comple- ment these data with gene expression analyses for Gata-1low mice of all ages, and to overcome the small sam- ple size (n=3 mice per group) in the RNAseq analyses, we
performed qPCR analyses for Ptpn1, Ptpn2, Ptpn6, Ptpn11, Ptpn12, and Ptprj. Indeed, in the gene expression analyses we observed distinct expression dynamics among gene expression of these PTP (Figure 5E-J). Overall, the data generated by qPCR from early fibrotic bone marrow of 10-month-old Gata-1low mice displayed pronounced bio- logical variances, possibly contributing to the lack of sig- nificance within the RNAseq data. Interestingly, Ptpn1 and Ptprj gene expression, analyzed by qPCR, showed decreased expression in pre-fibrotic bone marrow. There was an increased expression of Ptpn11 and Ptpn12 in early
ABC
H
DE
FG
Figure 7. Regulation of platelet-derived growth factor receptor β (PDGFRβ) signaling and proliferation of NIH-3T3 fibroblasts by T-cell protein tyrosine phosphatase (TC-PTP). (A) Immunoblot of Ptpn2 knock down (KD) and non-targeting (NT) control cells, untreated (–) and stimulated with 50 ng/mL PDGF-BB (+) for 5 minutes. (B) Densitometric analyses of TC-PTP, (C) pPDGFRb Y751, (D) pPDGFRb Y1021, (E) pPLCγ1 Y783, (F) pAKT S473, (G) pERK1/2 T202/Y204. (H) Proliferation curves of Ptpn2 KD and NT control cells cultured in 1% and 10% fetal bovine serum (FBS). *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 by analysis of variance with post hoc Tukey correction.
haematologica | 2020; 105(8)
2091