Expression of Hmga2 enhances activation of the TGF-β1 signaling pathway in Jak2^V617Fmouse BM and human megakaryoblastic SET-2 cells. (A) Total levels of TGF-β1 in the serum of WT-vector, WT-Hmga2, Jak2^VF/+-vector, and Jak2^VF/+-Hmga2 mice at 32 weeks after BMT were assessed by ELISA (n = 6-7). (B) Overexpression of HMGA2 increases the TGF-β1 mRNA expression in JAK2^V617F-positive megakaryoblastic SET-2 cells. (C) Lentiviral shRNA-mediated knockdown of HMGA2 decreases the TGF-β1 mRNA expression in SET-2 cells. The mRNA expression was assessed by RT-qPCR and normalized by GAPDH. Data from 4 independent experiments are shown in bar graphs as mean ± SEM. (D) HMGA2 ChIP followed by RT-qPCR showed binding of HMGA2 in the promoter of the TGF-β1 gene in SET-2 cells. Results from 3 independent experiments are presented as mean ± SEM in bar graphs. The RT-qPCR products were loaded onto 2% agarose gel. Representative picture from agarose gel is shown in the bottom panel. (E) Immunoblot analysis shows increased phosphorylation of SMAD2, SMAD3, AKT, p38 MAPK, and ERK1/2 in Jak2^VF/+-Hmga2 BM compared with Jak2^VF/+-vector BM. Total SMAD2, SMAD3, and AKT protein levels were also higher in Jak2^VF/+-Hmga2 BM compared with Jak2^VF/+-vector BM. However, total p38 MAPK and ERK1/2 levels were comparable. β-actin was used as a loading control. (F) Immunoblot analysis shows increased phosphorylation of SMAD2, SMAD3, AKT, p38 MAPK, and ERK1/2 in SET-2 cells overexpressing HMGA2 compared with vector-expressing SET-2 cells. β-actin was used as a loading control. *P < .05; **P < .005.