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Article Figures & Data

Figures

  • Figure 1.

    Expression of SHP1, NPM-ALK, JAK3, and STAT3 in 3 ALK+ALCL cell lines, Karpas 299, SUP-M2, and SU-DHL-1. Only SUP-M2 expressed SHP1, and this cell line also had relatively lower levels of pSTAT3, pJAK3, and JAK3 compared with the other 2 cell lines.

  • Figure 2.

    SHP1 expression and down-regulation of the JAK3/STAT3 pathway. (A) SHP1 expression induced down-regulation of the JAK3/STAT3 signaling pathway in SU-DHL-1 and Karpas 299 cells. SU-DHL1 cells transfected with pIRES2-EGFP or pIRES2-EGFP-SHP1 were sorted using flow cytometry and subjected to Western blot analysis 24 hours after gene transfection. The results showed substantial decreases in the protein expression of pSTAT3, pJAK3, and JAK3. In contrast, there were relatively few changes in the protein level of STAT3 between the 2 samples. Karpas 299 cells were transfected with pCI (empty vector) and pCI-SHP1, and subjected to Western blot analysis 24 hours after gene transfection. Similar to the SU-DHL-1 cells, Karpas 299 cells showed down-regulation of the JAK3/STAT3 signaling. (B) Modulation of STAT3 downstream targets as well as NPM-ALK in Karpas 299 and SU-DHL-1 cells after SHP1 gene transfection. SHP1 expression in Karpas 299 and SU-DHL-1 cells induced similar changes in the STAT3 downstream targets including bcl-2, mcl-1, and cyclin D3. Survivin was only slightly down-regulated. The protein level of NPM-ALK was also decreased. Cell lysates were prepared 24 hours after gene transfection. Cells transfected with pCI empty vector and pIRES2-EGFP served as negative controls for Karpas 299 and SU-DHL-1 cells, respectively. (C) Blockade of SHP1 expression using siRNA in SUP-M2. Inhibition of SHP1 in SU-DHL-1 cells using siRNA induced down-regulation of the expression of SHP1, with 200 pM more effective than 100 pM. There were increases in the expression of pSTAT3, pJAK3, JAK3, and NPM-ALK. One of the STAT3 downstream targets, cyclin D3, was also up-regulated. SUP-M2 cells transfected with the sense SHP1 siRNA served as negative controls.

  • Figure 3.

    Effect of MG-132. (A) MG-132 reversed the decrease of NPM-ALK and pJAK3 induced by SHP1. SU-DHL1 cells were transfected with pIRES2-EGFP-SHP1 and sorted based on GFP expression by flow cytometry. MG132 was then added 24 hours after gene transfection. The reduction in the levels of JAK3 and NPM-ALK induced by SHP1 was completely reversed by MG132 at 3 to 5 hours after the addition of MG132 to the cell culture. Cells transfected with pIRES2-EGFP (sorted based on GFP expression) served as negative controls. (B) MG132 did not induce up-regulation of JAK3, pSTAT3, and NPM-ALK in nontransfected SU-DHL cells. In contrast with the SHP1-transfected SU-DHL-1 cells, nontransfected SU-DHL-1 cells treated with MG132 for 3 to 5 hours showed no increase in the protein levels of NPM-ALK, pSTAT3, and JAK3.

  • Figure 4.

    A time-course experiment illustrating that SHP1 dephosphorylated JAK3 independent of down-regulation of the total JAK3 protein. SHP1 slowly increased its protein levels within the first 10 hours after SHP1 gene transfection into SU-DHL-1 cells. A decrease in pJAK3 was detectable at 4 hours, at which time no significant changes in the expression of JAK3 and NPM-ALK were observed.

  • Figure 5.

    SHP1 coimmunoprecipitated with NPM-ALK and JAK3. Coimmunoprecipitation studies indicated that SHP1 physically interacts with NPM-ALK and JAK3.

  • Figure 6.

    SHP1 induced G1cell cycle arrest and a decrease in the number of viable cells. Cell cycle analysis for SU-DHL1 cells transfected with pIRES2-EGFP and pIRES2-EGFP-SHP1. Cells were harvested 24 hours after gene transfection and subjected for cell sorting based on GFP expression. All GFP+ cells were then subjected to cell cycle analysis. SHP1 induced a significant increase in the G0/1 population, indicating cell cycle arrest.

  • Figure 7.

    SHP1 induced no significant apoptosis. In addition to the lack of an increase in the sub-G0/1 cell population, SHP1 expression in SU-DHL-1 cells also did not lead to detectable cleaved PARP product (arrow) and active caspase-3. Nontransfected SU-DHL-1 cells treated with different doses of doxorubicin served as positive controls.