DAPT decreases activated Notch protein, transcription of downstream effectors, T cell proliferation, and cytokines. (A,B) Lymph node cells were harvested from mice after 4 days of treatment with DAPT or control. Panel A depicts immunoblot of activated Notch protein (Notch 1C) showing significantly decreased activated protein in 2 representative treated animals compared with 2 controls. In addition, RNA was isolated to assess down-stream targets of Notch1C activated transcription, including Hes1 and Deltex1. (B-tubulin was used as internal control.) Panel B depicts RT-PCR of Hes1 and Deltex1, comparing a representative control and treated animal. Error bars represent SD. (C,D) CBA-lpr^cg mice were treated with DAPT or control for 3 months (5 treated; 3 control). At killing, lymph node cells, splenocytes, and thymocytes were harvested, supported in vitro, and stimulated with mitogen. After 24 hours, supernatants were collected and cytokines assessed by cytometric bead array. After 48 hours, proliferation was measured by MTT. Panel C depicts proliferation, comparing treated to control. Bars represent mean of the treated/control (untreated) cells (relative absorbance of triplicate cultures, as described in “T-cell functional assays”); error bars represent SD. Panel D depicts results of cytokine bead array, demonstrating elevated levels of IL-10 and MCP-1 and a marked and statistically significant decrease in treated animal. Error bars depict SEM. Similar results were found with MRL mice.