Microarray analysis reveals up-regulation of noncanonical Wnt signaling in CXCL12-mediated T-cell migration. (A) Primary T cells from 3 different donors were treated with human CXCL12 at 100 ng/mL for 12 hours. Cells from the same 3 donors were incubated in media only as a control. RNA was extracted from each sample and subjected to microarray analysis (6 arrays total). Genes with a 2.5-fold cutoff (either up or down) were considered significantly changed. Genes involved in noncanonical Wnt signaling (arrows) were found to be significantly up-regulated (> 2.5-fold increase, P < .05) in the CXCL12-treated T-cell groups. (B) Primary T cells from 4 additional donors were treated with human CXCL12 at 100 ng/mL for 12 hours. Cells from these 4 donors were incubated in media only as a control. RNA was extracted and cDNA was used to perform real-time PCR analysis using the Open Biosystems Superarray pathway reverse transcription–PCR kits. Superarray reverse transcription–PCR analysis confirms that gene members of the noncanonical Wnt signaling pathway are up-regulated (B), whereas members of the canonical Wnt signaling pathway are down-regulated (C). All genes are normalized to actin. Interestingly, the only gene up-regulated in the canonical Wnt signaling pathway is the Wnt inhibitory factor-1 (WIF1), an inhibitor of canonical Wnt signaling. To validate the RNA results, Western blot analysis was performed. (D) Primary human T cells (pool of 3 donors) were treated with 100 ng/mL CXCL12 for the times indicated, then examined by Western blot analysis for β-catenin, Wnt5A, and Fzd2 expression. These data demonstrate that β-catenin is decreased by CXCL12 treatment, but Wnt5A and Fzd2 expression are increased (E). (F) Wnt5A up-regulation is specific to CXCL12, as Western blot analysis demonstrates that CCL19 (100 ng/mL, 12 hours) does not increase Wnt5A expression. Western blots were repeated 3 times, and a representative blot is shown.