Effect of deletion of SPTLC1 on gene expression and differentiation of mouse Th17 cells in vitro
Th17 cells are implicated in autoimmune diseases and several metabolic processes are shown to be important for their development and function. However, the role of de novo sphingolipid biosynthesis in their differentiation and function is less known. To investigate the role of SPTLC1(major subunit of serine palmitoyl transferase enzyme (SPT) that catalyzes the first and rate limiting step of de novo sphingolipid synthesis) in mouse Th17 cell differentiation, we have activated the WT and SPTLC1 defecient (KO) naïve CD4+ T cells, isolated  from mouse spleen and lymph nodes, in Th17 differentiating condition in the precence and absence of NAC ( N-acetyl cysteine) and 3-KDS (3-ketodihydrosphingosine). Overall design: To investigate the role of SPTLC1 in Th17 differentiation, total RNA was extracted for each of the 3 biological replicates from WT and KO naive CD4+ T cells and Th17 cells. To understand the specificity of the de novo sphingolipid biosynthetic pathway in the observed effect KO Th17 cells, RNA was isolated from WT and KO naive CD4+T cells cultured under Th17 polarizing condition in the presence and absense of 3-KDS (product of SPT enzyme). Further, to understand role of ROS in the observed defect in KO Th17 cells,  RNA was isolated from WT and KO naive CD4+T cells cultured under Th17 polarizing condition in the presence and absense of 3-KDS (product of SPT enzyme)