Expression data from activated and non-activated NTAL knockout, wild type and knockdown bone marrow derived mast cells

Non-T cell activation linker (NTAL, also called Lab or LAT2) is a transmembrane adaptor protein involved in the high affinity IgE receptor (FcεRI) signaling in mast cells. Studies with bone marrow-derived mast cells (BMMCs) from NTAL-/- mice indicated that NTAL is a negative regulator of FcεRI signaling. In contrast, depletion of NTAL by RNA interference in human or rat mast cells suggested its positive regulatory role. To determine whether compensatory developmental alterations are responsible for the observed discrepancies, we compared FcεRI-mediated signaling events in BMMCs isolated from NTAL-/- mice or BMMCs of wild type in which NTAL expression was decreased by RNA interference. We found that when compared to corresponding controls, NTAL-deficient cells, both knockouts (KOs) and knockdowns (KDs), exhibited enhanced degranulation, calcium mobilization, tyrosine phosphorylation of linker for activation of T cells (LAT) and ERK, depolymerization of filamentous actin and chemotaxis. These data indicate that NTAL in mouse BMMCs is a negative regulator of FcεRI signaling, independently of compensatory developmental alterations. To gain more insight into NTAL function, we examined gene expression profiles of resting and antigen-activated BMMCs with NTAL KO or KD and corresponding controls and identified several genes that were more than 1.8-fold differentially expressed in resting and FcεRI-activated NTAL-deficient cells, when compared to wild-type controls. We provide evidence that at least some of these genes could be involved in regulation of cholesterol-dependent events in chemotaxis towards antigen. Two types of NTAL-deficient bone marrow derived mast cells (BMMCs) were used (1) cells isolated from NTAL KO mice and (2) NTAL KD cells prepared by lentiviral infection of the NTAL shRNA into WT cells. Activated and nonactivated NTAL-deficient mast cells were compared with the WT cells and the WT cells infected with the empty vector pLKO.1 used as a negative control to NTAL KDs. Three biological replicates of each sample were analyzed. One of the 24 samples, KO 2_2 h, failed during processing and was removed.