DM does not induce intracellular Ca⁺⁺ but directly induces and augments CXCL12-induced actin polymerization and Rac1 and Vav activation. (A) Jurkat cells were loaded with 2 μM Fura-2 after which the levels of intracellular calcium were assessed using a spectrofluorimeter. Fluorescence was monitored at λ_ex1 = 340 nm, λ_ex2 = 380 nm, and λ_em = 510 nm. At all times, the baseline was maintained for 300 seconds followed by addition of CXCL12 (100 ng/mL) or DM (1 μM) in the cuvette. A second addition of CXCL12 was performed at 700 seconds to assess receptor desensitization. Ionomycin was added at 1000 seconds of all samples to assess Fura load and cellular viability and responsiveness. In the DM-treated groups, the cells were pretreated with 1 μM DM for 5 minutes to examine its direct effects on cellular calcium levels and on CXCL12-induced intracellular calcium mobilization. (B) Primary T cells were treated with CXCL12 (100 ng/mL) and DM (1 μM) or the combination for 5 minutes and were fixed followed by staining for polymerized actin using phalloidin labeled with Alexa Fluor 488. The cells were subsequently cytospinned on a slide coated with poly-L-lysine and then mounted with vectashield mounting media and examined using a Zeiss Axiovert S100 microscope (Carl Zeiss, Thornwood, NY) using a Spot Enhanced EN-1400 CCD camera and analyzed by Spot Advanced software (Diagnostic Instruments, Sterling Heights, MI). The fluorescence image was acquired using the green filter for Alexa Fluor 488; 1, 2, 3, and 4 in the panel indicated the control, CXCL12, DM, and combined treatment, respectively. (C) Primary human T cells were treated with CXCL12 (100 ng/mL), DM (1 μM), or the combination for 5 minutes. The cells were subsequently fixed with 2% paraformaldehyde solution for 1 hour followed by incubation with phalloidin-Alexa Fluor 488. Data acquisition was performed by forward scatter versus side scatter. MFI indicates the mean fluorescence intensity. (D) Primary human T cells were treated with CXCL12 (100 ng/mL), DM (1 μM), or the combination for 5 minutes and then lysed with RIPA buffer. Rac1 was immunoprecipitated by Pak1 using the kit supplied by Chemicon International followed by separation in sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then immunoblotted. The densitometry of each band was performed and each value was first normalized against the total Pak1 and then presented as fold change over control of activated Rac1 over Pak1. (E) Similar to Rac1, the same procedure was used for Vav1 and Vav2 except that the immunoprecipitation was performed by assessing total Vav1 and Vav2, and the densitometric value of each phospho-Vav1 and Vav2 band was initially normalized against the total Vav1 or Vav2 (P/T) and then presented as fold change over control.