STAT3 and STAT5 function as oncogenic effectors of Syk. KG-1 cells were subjected to retroviral transduction with IRES-EGFP vectors inducing stable expression of EGFP, caSTAT3 and EGFP, or caSTAT5 and EGFP. (A) After fluorescence-activated cell sorting for EGFP, the percentage of GFP-expressing transduced KG-1 cells was monitored by flow cytometry. (B) Syk was knocked down by shRNA treatment in the sorted KG-1 cell batches that were enriched for EGFP-expressing cells. Expression of STAT3, STAT5, Syk, Socs3, and actin was monitored by immunoblot analyses 24 hours after lentiviral shRNA treatment. The transduced cell batches were left untreated (C) or were treated with the Syk inhibitor Bay 61-3606 at a final concentration of 250 nM for up to 7 days (D). (E) The transduced cell batches were left untreated or were treated with shRNAs specific for Syk. After confirmation of successful knockdown of Syk, cells were cultured for up to 7 days. The proportion of EGFP-positive cells was analyzed by flow cytometry (C-E, left panels). Absolute cell numbers are shown in the right panels. Wild-type (WT) KG-1 cells were used as the negative control in all flow cytometric measurements (data not shown).