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. Author manuscript; available in PMC: 2018 Mar 7.
Published in final edited form as: Cell Metab. 2017 Mar 7;25(3):686–697. doi: 10.1016/j.cmet.2017.02.010

Figure 4. Effects of ACSL5 and DGAT2 Modulation on Ceramide and Acylceramide Levels.

Figure 4

Species specific (A) and total (B) ceramide content of cells stably expressing vector control or HA-ACSL5 was measured by LC/MS.

(C and D) C16-acylceramide levels (Mild base sensitive) of cells stably expressing vector control or ACSL5 (C) and cells transfected with Scr (control) or ACSL5 siRNAs

(D) was measured by LC/MS.

(E) Sphingosine and ceramide levels of cells transfected with Scr (Control) or DGAT2 siRNAs was measured using LC/MS.

(F and G) C16-acylceramide levels (Mild base sensitive) of cells transfected with Scr (control) or DGAT2 siRNAs (F) and cells stably expressing LacZ (control) or DGAT2

(G) was measured by LC/MS.

(H) In vitro activity of DGAT2 for generation of acylceramide was determined as described in Materials and Methods. O-acyl-NBD-C12-ceramide band is indicated with an arrow.

(I) In vitro activity of wild type (WT) and H163A mutant (Mut) DGAT2 for generation of acylceramide was determined.

(J) Microsomes from LacZ or DGAT2 expressing cells were incubated with PF-06424439 and their in vitro ceramide acyltransferase activities were determined.

Results are expressed as mean of three independent experiments with error bars representing ± SEM. *p <0.05 and ** p< 0.01.