Serine pSTAT3 initiates transcription of STAT3-regulated genes in CLL cells. CLL cells were infected with a lentivirus harboring GFP-STAT3-shRNA or with lentivirus GFP-empty vector. (A) RT-PCR results of mRNA levels of STAT3 and the STAT3-regulated genes Bcl2, Pim1, Bcl-X_L, Cyclin D1, p21 (Waf1), and c-Myc in CLL cells (CLL 26) that were infected by lentivirus harboring GFP-STAT3-shRNA for 24, 48, and 72 hours. (B) RT-PCR results of mRNA levels of STAT1, STAT3, and STAT5 in CLL cells (CLL 26) that were infected by lentivirus harboring GFP-STAT3-shRNA for 24 and 72 hours. The RT-PCR results are shown as fold change (decrease or increase) relative to the mRNA levels of the STAT3-regulated genes in CLL cells that were infected with lentiviral GFP-empty vector. (C) RT-PCR results of mRNA levels of the STAT3-regulated genes STAT3, Bcl2, Pim1, Bcl-X_L, p21 (Waf1), and c-Myc in CLL cells (CLL 15, 19, 24, and 27). Changes in mRNA levels (mean ± SD) are shown. As in the previous experiment, infection with empty virus did not significantly affect STAT3-regulated gene levels. (D) Left panel: Infection efficiency analyzed by flow cytometry of control and GFP⁺ empty virus– and STAT3-shRNA–infected CLL cells (CLL 26). Right panel: Western blot of control, empty vector- and STAT3 shRNA-infected CLL cells. Forty-eight hours after infection with STAT3, shRNA down-regulated STAT3 protein levels by 50%. (E) Flow cytometric analysis of PI^+/− and annexin V⁺ cells 48 and 72 hours after infection with GFP-STAT3-shRNA or empty vector. The percentages of early apoptotic cells are shown in the bottom right corners and of late apoptotic (PI⁻ and annexin V⁺) cells in the top right corners.