Volume 22, Issue 12, Pages 1662-1670 (December 2015) Differential Regulation of Specific Sphingolipids in Colon Cancer Cells during Staurosporine-Induced Apoptosis  Virginia del Solar, Darleny Y. Lizardo, Nasi Li, Jerod J. Hurst, Christopher J. Brais, G. Ekin Atilla-Gokcumen  Chemistry & Biology  Volume 22, Issue 12, Pages 1662-1670 (December 2015) DOI: 10.1016/j.chembiol.2015.11.004 Copyright © 2015 Elsevier Ltd Terms and Conditions

Chemistry & Biology 2015 22, 1662-1670DOI: (10. 1016/j. chembiol. 2015 Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 1 Immunofluorescence Detection of Apoptotic Cells and PCA of Lipid Composition of Staurosporine-Treated and Non-treated HCT-116 Cells (A) Representative control and staurosporine-treated apoptotic HCT-116 cells. Apopxin (marker for apoptotic cells) and calcein (marker for live cells) are shown in green and red, respectively. Scale bar, 25 μm. (B) PCA of 16-hr staurosporine-treated (blue), 5-hr staurosporine-treated (red), and control (black) HCT-116 cells. We observed distinct clustering among 16-hr staurosporine-treated and the other two conditions without any a priori input, suggesting major global changes in lipid composition after 16 hr of treatment. PCA plot was generated in MPP 12.6.1 (Agilent Technologies). Chemistry & Biology 2015 22, 1662-1670DOI: (10.1016/j.chembiol.2015.11.004) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 2 Sphingolipids that Accumulate Significantly during Staurosporine-Induced Apoptosis in HCT-116 Cells Based on Untargeted Lipid Profiling Results are based on three independent profiling experiments, n = 5 for each profiling experiment. p ≤ 0.05. The position of the double bond in acyl chains could not be determined. Chemistry & Biology 2015 22, 1662-1670DOI: (10.1016/j.chembiol.2015.11.004) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 3 Simplified Scheme of Sphingolipid Biosynthesis The scheme is modified from Wennekes et al. (2009). De novo ceramide biosynthetic and sphingomyelin hydrolysis pathways are shown in red and green, respectively. Lipids that accumulate during staurosporine-induced apoptosis in HCT-116 are shown in red (dihydroceramides), blue (ceramides), and green (sphingomyelins). The expression levels of CERSs are higher in apoptotic cells. The activities of SMases are lower, whereas the activity of SMS does not change during apoptosis. Chemistry & Biology 2015 22, 1662-1670DOI: (10.1016/j.chembiol.2015.11.004) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 4 Expression Levels of CERSs and Enzymatic Activity for SMase and SMS in HCT-116 Cells (A) Different ceramide synthases (CERS1–6) and their products based on acyl chain specificity. Sphinganine backbone and different fatty acids are shown in red and black, respectively. (B) Changes in the expression of different CERSs in 16-hr staurosporine-treated HCT-116. mRNA levels of CERS2, CERS4, CERS5, and CERS6 were measured and normalized based on the expression level of HRPT1. Significant increases in CERS2, CERS4, and CERS5 were observed with staurosporine treatment. Data from three independent experiments (n = 9) are shown as means ± SD. (∗∗p < 0.01). (C) Activities of nSMase, aSMase and SMS were measured in control and apoptotic cells. There was a significant decrease in nSMase and aSMase, whereas SMS activity did not change during apoptosis. Data from three independent experiments (n = 15) are shown as means ± SD. (∗∗p < 0.01; ∗∗∗p < 0.001). Chemistry & Biology 2015 22, 1662-1670DOI: (10.1016/j.chembiol.2015.11.004) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 5 Differences in Ceramide, Sphingomyelins, and CERS Expression Levels in HCT-116 and CCD-112 and Cell Viability after Add-Back Experiments (A) Fold differences in ceramide (Cer) and sphingomyelin (SM) levels in CCD-112 and HCT-116 cells. For each ceramide and sphingomyelin, fold difference is determined by [AbundanceCCD-112]/[AbundanceHCT-116]. Abundance is the total ion count for a given ion. Lipid composition is normalized based on protein concentration and internal standards. CCD-112 cells produce similar levels of ceramides but higher levels of sphingomyelins compared with HCT-116. Dotted line indicates no difference (fold difference = 1). Data from two independent experiments (n = 10) are shown as means ± SD. (B) Expression levels of CERS2, CERS4, CERS5, and CERS6 were compared in HCT-116 and CCD-112 cells. mRNA levels of CERS2, CERS4, CERS5, and CERS6 were measured and normalized based on the expression level of HRPT1. CCD-112 expresses significantly higher levels of CERS2, CERS4, and CERS5. Data from three independent experiments (n = 9) are shown as means ± SD. ∗p < 0.05, ∗∗p < 0.01. (C) Cell viability in HCT-116 after C16-ceramide (white bars) and C16-dihydroceramide (gray bars) treatment. Data from three independent experiments (n = 15) are shown as means ± SD. (D) Cell viability in CCD-112 after C16-ceramide (white bars) and C16-dihydroceramide (gray bars) treatment. Data from three independent experiments (n = 15) are shown as means ± SD. Chemistry & Biology 2015 22, 1662-1670DOI: (10.1016/j.chembiol.2015.11.004) Copyright © 2015 Elsevier Ltd Terms and Conditions