Figure 2. Shh treatment influences DN32 iNKT cell growth, viability, and cytokine production.

DN32 cells were seeded at a concentration of 1 × 10⁵ cells per well, and incubated with recombinant Shh protein (10 to 1000 ng/ml) for 72 h. Control wells were treated with an equal volume of vehicle (Shh 0 ng/ml) for the same treatment period. Triplicate wells were assayed in each of the experiments and each experiment was performed in duplicate. At the end of the treatment period, A) growth (cell number) was assessed using the CCK-8 assay, B) apoptotic activity was evaluated by measuring caspase 3/7 activity, C) cytokine secretion (IFNγ , IL4, IL13, IL10) was measured by ELISA. As maximal cytokine protein production was observed when DN32 were treated with Shh 100 ng/ml, subsequent studies to assess mRNA expression of cytokines and suppressors of cytokine signaling (SOCS) were conducted with Shh doses ranging between 0 to 100 ng/ml. D) Cytokine mRNA gene expressions were determined by qRT-PCR analysis. E) SOCS-2 and F) SOCS-3 mRNA levels were quantified by Q RT PCR. Results are expressed as fold change (± SEM) relative to cells that were treated with vehicle (0 ng/ml Shh). *p<0.05 vs. vehicle (Shh 0 ng/ml) using Student’ s t-test.