BCL6 inhibits expression of the endogenous STAT3 gene in DLBCL. (A) BCL6 and MTA3 bind to the STAT3 promoter region in vivo. ChIP assays were performed in Ly1 cells. PCR products were amplified from chromatin fragments immunoprecipitated with either anti-BCL6, anti-MTA3, or control rabbit IgG antibodies. The PCR primers flank either site B or the negative control +20-kb site (see arrows in Figure 4A). H₂O, negative control PCR without any template; input, purified total genomic DNA before precipitation. (B) Forced expression of BCL6 suppresses endogenous STAT3. Ly10 cells were transiently transfected with the indicated expression plasmids. Western blot was performed to analyze expression of BCL6 and MTA3 12 hours after transfection, and total STAT3 24 hours after transfection. Similar results were obtained from Ly3 cells (not shown). (C) RNAi-mediated BCL6 knockdown increased STAT3 protein expression. Control (ctrl) and 2 different BCL6 siRNA oligos (nos. 1 and 2) were transiently transfected into Val cells, and cell lysates were prepared at either 24 or 48 hours for protein analysis. In lanes 7 to 10, cells that have been transfected with oligos for 45 hours were either left alone (lanes 7 and 8) or exposed to PMA (20 ng/mL) and ionomycin (0.3 μM) (P + I, lanes 9 and 10) for another 24 hours before harvesting. In lane 11, Ly3 lysates were loaded for comparison. BCL6, total STAT3, and PY-STAT3 proteins were examined by Western blot analysis. GAPDH levels were shown as loading control. Vertical lines have been inserted to indicate a repositioned gel lane.