Shp2 modulates Kit expression through Gata2. (A, B) qRT-PCR of Kit and Kit regulatory transcription factors Scl, Sp1, Gata2, and Gata1 mRNA demonstrated consistent suppression of Kit and Gata2 transcription in primary Shp2^Δ/Δ lineage⁻ cells and Shp2 knockdown EML cells. β-actin was used as a reference gene for normalization. (C) Shp2 knockdown repressed Kit promoter activity. Constructs with Kit regulatory elements fusion to GFP reporter were transfected into HEK293 cells. Shp2 knockdown by specific siRNA lead to GFP down-regulation in both p13kit and p70kit transfectants but not GFP vector transfectant. Erk was used as loading control. (D) Enforced expression of flag-tagged wild-type (WT) and dominant-active (DA) Shp2-enhanced, dominant-negative (DN) Shp2 suppressed Kit promoter activity, with no effect for truncated Shp2 containing the 2 SH2 domains. Erk or Tubulin was used as loading control. (E) Signaling modification in Shp2 knockdown EML cells elicited by SCF. Tubulin was used as loading control. (F) Treatment of EML cells with PI3K inhibitor LY294004, MEK inhibitor U0126, or a low dose (500 nm) of LY294004, U0126, and STAT3 inhibitor AG490 led to down-regulation of Kit expression on cell surface. (G) Shp2 knockdown in EML cells suppressed Gata2 protein expression. Tubulin was used as loading control. (H) Ectopic expression of Gata2 rescued Kit down-regulation in Shp2 knockdown EML cells. (I) Quantitative analysis of Gata2 binding to the −114 kb, +5 kb, and promoter region of Kit gene by the CHIP assay. Binding of Gata2 to Kit promoter but not −114 kb or +5 kb region was significantly reduced in Shp2 knockdown EML cells. *P < .05; error bars are SD. (J) Kit staining profile of wt lin⁻Sca1⁺CD48⁻CD150⁺ cells, Shp2^Δ/Δ lin⁻Sca1⁺CD48⁻CD150⁺ cells, Shp2^Δ/Δ/Pten^Δ/Δ lin⁻Sca1⁺CD48⁻CD150⁺ cells. At least 3 animals were examined in each group. Representative FACS plot was shown; see also supplemental Figure 5.