Control of Tcf4 expression by Flt3L-responsive STAT3. (A) Hematopoietic-Stat3^Δ/Δ mice and Stat3^+/+ control animals were treated by Flt3L HGT or pORF HGT vector as indicated. CDPs were isolated from BM after 2 days and analyzed for Tcf4 expression by quantitative PCR, using primers listed in supplemental Table 1. Tcf4 mRNA was normalized to Rpl13a mRNA amounts. Results represent average values from 2 independent experiments. Error bars represent SEM of average values. P values of indicated comparisons are shown. (B) Purified CDPs from hematopoietic-Stat3^Δ/Δ and Stat3^+/+ controls were cultured ex vivo with Flt3L for 24 hours, 72 hours, or left untreated. Tcf4 expression was analyzed by quantitative PCR as described in panel A. Results are presented as the fold induction of Flt3L-treated samples relative to untreated samples. Results represent average values from 3 independent experiments. (C) D2SC/mFlt3 cells were transfected with the PGL4.12/Tcf4 reporter construct or empty vector, together with phRL-TK-Renilla and plasmids expressing WT STAT3, a STAT3 isoform defective in DNA binding (DN) or a STAT3 isoform lacking the C-terminal transactivation domain (TAD). Cells were treated with Flt3L for 2 hours or left unstimulated. Results are expressed as normalized values (normalized values = RLU from Flt3L-treated cells/RLU from untreated cells). Data represent the mean ± SEM of 3 independent experiments. (D) EMSAs were performed with nuclear extracts isolated from D2SC/mFlt3 cells after stimulation with or without Flt3L for 30 minutes, using ³²P-labeled oligonucleotide probes corresponding to the STATx consensus site in Tcf4, with or without competitor oligonucleotides (T indicates Tcf4 probe; S3, STAT3 binding site in murine Socs3 promoter; and m, mutant Tcf4 probe), or anti-STAT3 antibody (☆ represents STAT3/DNA complexes; and ★, antibody supershifted STAT3/DNA complexes), as indicated (left). Control EMSAs were performed with a consensus STAT3 probe from the Socs3 promoter as indicated (right). (E) ChIPs were performed with D2SC/mFlt3 cells treated with or without Flt3L for 1 hour, using anti-STAT3 or IgG control antibody, as indicated. Purified DNA from immunoprecipitated samples was analyzed by quantitative PCR. Results were normalized to total cell lysate (input). (D-E) Results represent data from 3 independent experiments.