Sphingolipids Control Lifespan Figure 4. Myriocin or Dox treatment reduces sphingolipid levels. (A) LCBs and LCBPs were quantified in cells grown from an A600 nm of 0.005 to 1 in the presence of absence of Dox (300 ng/ml). Average values for triplicate cultures of tetO7-LCB1 (RCD956) cells are shown (* p,0.05, ** p,0.01, *** p,0.001, No Dox vs Dox-treated). (B) LCBs and LCBPs were quantified in DBY746 cells treated or not treated with Myr (400 ng/ml) grown the same way as in (A). Average values for triplicate cultures are shown (* p,0.05, ** p,0.01, *** p,0.001, No Myr vs Myr-treated). (C) Complex sphingolipids (IPCs, MIPCs and M(IP)2Cs) in untreated and Dox-treated RCD956 cells were grown as in (A) in the presence of [3H]-myoinositol and cpm were quantified by scanning the thin-layer chromatogram on a Bioscan apparatus. Glycerol –P-inositol represents the deacylation product of phosphatidylinositol and serves as an internal control. (D) Average cpm +/2 SD for [3H]-labeled lipids from three cultures analyzed as in (C). (E) Average cpm +/2 SD for [3H]-labeled lipids from three cultures of DBY746 cells treated or not treated with Myr (400 ng/ml). Cells were grown and analyzed as in (C). doi:10.1371/journal.pgen.1002493.g004 PLoS Genetics | www.plosgenetics.org 8 February 2012 | Volume 8 | Issue 2 | e1002493