Sp1 DNA-binding activity is high in WM cells and affects tumor growth and viability. (A) Nuclear extracts from MWCL1, BCWM1, and normal CD19⁺ cells were analyzed using TF activation ELISA array. TF activation profile differences in cancer and normal cells were quantitatively analyzed and compared. TFIID was used as loading control. The radar chart displays fold change values (from 0 to 7) in MWCL1 and BCWM1 cells relative to CD19⁺ normal cells. (B) Nuclear extracts from WM and low-grade lymphoma cell lines were analyzed for Sp1 activity using the Sp1 TF ELISA kit, which measures Sp1 DNA binding activity. To analyze the specificity of the DNA-binding complexes, cold competition was performed (data not shown). Absorbance was obtained with a spectrophotometer at 450 nm and presented as optical density. Blue, red, and green lines show mean Sp1 binding activity of WM, low-grade lymphoma, and normal CD19⁺ cells, respectively. (C) Equal amounts of nuclear and cytoplasmic protein extracts from MWCL1 (MW), BCWM.1 (BC), WSU-WM (WS), RL, MEC, and CD19⁺ normal cells from 2 healthy donors were subjected to WB analysis using anti-Sp1 ab. p84, and GAPDH were used as loading controls for nuclear and cytoplasmic fractions, respectively. (D) BCWM1 and MWCL1 cell lines were transfected using different concentrations of TranSilent Human Sp1 siRNA or scrambled siRNA (Scr). Cell lysates were obtained 48 hours after transfection and subjected to WB analysis to assess decrease in the Sp1 protein expression posttransfection using anti-Sp1 and GAPDH abs. WB analyses confirmed reduction in Sp1 protein level following transient transfection of WM cells with Sp1 siRNA compared with cells transfected with control Scr. (E) The effect of Sp1 knockdown on cell survival in WM cells transfected with Sp1 or control siRNA were assessed by CellTiter Glo assay and presented as change relative to control cells. (F) WM cell lines transfected with 2 µM of Sp1 siRNA or control siRNA were cultured in the absence or presence of BMSC for 24 hours. Tumor cell growth was evaluated by [³H]thymidine uptake and presented as a percentage of cell growth compared with Scr.