Inhibition of Sp1 activity impacts NF-κB and STAT3 pathways in WM cells. (A) Nuclear extracts from Sp1 knockdown or TMP-treated MWCL1 cells were analyzed for TF activation using a TF profiling array. Relative fold changes from corresponding control are plotted. (B) BCMW1 cells were electroporated with control or Sp1 expression vector, NF-κB luciferase reporter plasmid, and pRL-TK to normalize for different transfection efficiencies; following electroporation, cells were treated with vehicle or 10 µM TMP, and 24 hours later luminescence was measured using the Dual-Luciferase assay kit and the GloMax microplate luminometer. Results are expressed as a percentage of Firefly/Renilla ratio of control-transfected cells. Whole lysates from these transfected cells were immunoblotted using anti-Sp1 and anti-α-tubulin (Santa Cruz Biotechnology) antibodies. (C) BCWM.1 cells were cultured with TMP (10 µM) for 24 hours, and then TNF-α was added for the last 20 minutes. NF-κB p65 TF binding to its consensus sequence on the plate-bound oligonucleotide was analyzed in nuclear extracts. The results represent means ± SD of triplicate experiments. (D) Whole cell lysates from Scr, or Sp1-specific siRNA- or control, or TMP-treated BCWM.1 cells were subjected to WB using anti–p-NF-κB p65, -NF-κB p65, and -GAPDH antibodies. (E) BCWM1 cells were treated with IL-6 with and without TMP and assessed by WB analysis using anti–p-STAT3, -STAT3, and -GAPDH antibodies.