RT-PCR analysis of HOXB9 after stimulation of HL cell lines. (A) HDLM-2 and KM-H2 were treated with 200 μM genistein and 14 mM NFκB inhibitor for 12 hours. In contrast to HDLM-2, genistein treatment reduced HOXB9 expression in KM-H2 cells, whereas NFκB inhibitor showed no effect. Antisense oligonucleotide (AS) treatment directed against E2F3A in SUP-HD1 and KM-H2 cells affected HOXB9 reduction relative to control oligonucleotide, indicating an activatory influence for this protein. (B) HDLM-2 and L-428 cells were treated with the following inhibitors of signaling pathways for 12 hours: 1 μM wortmannin (PI3K), 100 μM AG490 (JAK/STAT), 50 μM PD98059, and 100 μM SB202190 (ERK1/2 MAPK). HOXB9 showed reduction in HDLM-2 cells only, following MAPK pathway inhibition, indicating stimulation by this pathway. (C) KM-H2 cells were treated with 10 μg/mL inhibitory antibody directed against FGF2. HDLM-2 and L-540 cells were stimulated with 10 ng/mL recombinant FGF2 protein. HOXB9 expression decreased in KM-H2 and increased in HDLM-2 and L-540 cells. Data indicate an activatory influence of FGF2 on HOXB9 expression. (D) Influence of PKC on HOXB9 expression was analyzed by treating HDLM-2 and KM-H2 cells with a PKC activator TPA (100 nM) or PKC inhibitor calphostin C. Data show the absence of PKC activity in both cell lines. PKC activation reduced HOXB9 expression in KM-H2 but not in HDLM-2. NTC indicates no template control. For cDNA control the expression of ets variant gene 6 (TEL) or UBF was analyzed.