Fig. 3. Extracellular Ca²⁺ influx is essential but not sufficient for BCR-mediated SRF activation in WT DT40 cells. (A) BCR-mediated SRF activation is abrogated by chelating extracellular Ca²⁺ with EGTA. SRF transcriptional activity was determined in WT DT40 cells treated with or without anti-IgM in normal Ca²⁺ concentration, lacking extracellular Ca²⁺ (by adding EGTA) or in the presence of EGTA and reconstituted with Ca²⁺ (equimolar EGTA and Ca²⁺ concentrations). *P < 0.05 versus control without EGTA. (B) NiCl₂ blocks BCR-induced intracellular Ca²⁺ elevation. Intracellular Ca²⁺ response induced by anti-IgM stimulation with (bold line) or without (thin line) 1 mM NiCl₂ added at the indicated time in WT DT40 cells. The results are representative of three independent experiments. (C) NiCl₂ inhibits BCR-induced SRF activation. SRF transcriptional activity was determined in WT DT40 cells stimulated with anti-IgM in the absence or presence of the indicated concentrations of NiCl₂. The SRF–luciferase activity is shown as % activation (0 and 100% activation refer to no stimulation or stimulation with anti-IgM in normal medium, respectively, and are the mean ± SD of two independent experiments each performed in triplicate). (D) Intracellular Ca²⁺ elevation is not sufficient for SRF activation. SRF transcriptional activity was determined in WT DT40 cells treated with the indicated concentrations of ionomycin before harvesting for luciferase assay.