1. Ceramide Is Metabolized to Acylceramide and Stored in Lipid Droplets
Can E. Senkal, Mohamed F. Salama, Ashley J. Snider, Janet J. Allopenna, Nadia A. Rana, Antonius Koller, Yusuf A. Hannun, Lina M. Obeid 
Cell Metabolism 
Volume 25, Issue 3, Pages (March 2017)
DOI: /j.cmet
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2. Cell Metabolism 2017 25, 686-697DOI: (10.1016/j.cmet.2017.02.010)
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3. Figure 1 Determination of CerS-ACSL5 Association
(A) FLAG-tagged CerS1, 2, 4, 5, and 6 were immunoprecipitated after lysis in digitonin-containing buffer, and interactions of ACSL1 and ACSL5 with CerS was determined by western blotting.
(B) Interactions of ACSL5 and CerS were determined as in (A) using Triton X-100-containing lysis buffer.
(C) HA-tagged ACSL5 was expressed in cells stably transfected with FLAG-tagged CerSs. ACSL5-CerS interaction was determined by western blotting with anti-FLAG antibody after immunoprecipitation (IP) with anti-HA antibody.
(D) Co-localization of HA-ACSL5 with FLAG-CerS6 and calnexin was imaged by confocal microscopy.
(E) FLAG-tagged CerS and HA-ACSL5 interaction was identified using proximity ligation assay (PLA).
(F) Interaction of endogenous CerS6 with ACSL5 was detected by IP with anti-CerS6 or anti-ACSL5 antibodies followed by western blotting after control (Scr), CerS6, or ACSL5 siRNA transfections. Arrows indicate specific bands. Single and double asterisks indicate light and heavy chains of IP antibodies, respectively. Isotype-matched IgG was used as negative control.
Data are representative of three independent experiments.
Cell Metabolism  , DOI: ( /j.cmet )
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4. Figure 2
Identification of Domains in ACSL5 Required for CerS-ACSL5 Association
(A) Left: cellular localization of wild-type and mutants of ACSL5 was imaged. Right: diagrams of ACSL5 protein with domains that were modified. N-terminal, amino acids 1–66; TM, putative transmembrane domain; CNM1, common no motif 1; CNM2, common no motif 2; domain I, F316; AMP binding domain.
(B) Levels of wild-type and ACSL5 mutants determined by western blotting using anti-HA antibody (left panel). Association of wild-type and mutants of HA-tagged ACSL5 with FLAG-tagged CerS1 was determined by anti-FLAG IP and western blotting using anti-HA antibody (right panel). HC and LC, heavy and light chains of IP antibody. Arrow, FLAG-CerS1.
(C) Diagram of HA-tagged chimeric protein (N+TM-GFP) containing the first 88 amino acids of ACSL5 (N+TM) upstream of eGFP.
(D) Interaction of HA-tagged N+TM-GFP with V5-tagged CerS1 was identified by anti-V5 IP and western blotting with anti-HA antibody.
(E) Interaction of FLAG-CerS1 with N+TM-GFP and ER-GFP was determined by anti-FLAG IP and western blotting with anti-GFP antibody.
Data are representative of three independent experiments.
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5. Figure 3 Silencing of ACSLs Induces Ceramide Accumulation
(A) Cellular sphingosine and ceramide levels were measured by LC-MS after ACSL5 silencing. ACSL5 abundance after siRNA transfections (inset).
(B) Dihydroceramide levels were measured in cells transfected with Scr (control) or ACSL5 siRNAs.
(C) Sphingosine and ceramide levels were measured in cells transfected with All Star (control), ACSL1, or ACSL5 siRNAs individually. Downregulation of ACSL1 and ACSL5 was confirmed by western blotting (inset).
(D and E) Total sphingomyelin (D) and total ceramide levels (E) of cells transfected with indicated siRNAs and treated with PBS (control), fumonisin B1 (FB1), or myriocin (Myr) were measured by LC-MS.
Results are expressed as mean of three independent experiments with error bars representing ± SEM. ∗p < 0.05 and ∗∗p < 0.01.
Cell Metabolism  , DOI: ( /j.cmet )
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6. Figure 4
Effects of ACSL5 and DGAT2 Modulation on Ceramide and Acylceramide Levels
(A and B) 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) were measured by LC-MS.
(E) Sphingosine and ceramide levels of cells transfected with Scr (control) or DGAT2 siRNAs were 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) were measured by LC-MS.
(H) In vitro activity of DGAT2 for generation of acylceramide was determined as described in the Experimental Procedures. O-acyl-NBD-C12-ceramide band is indicated with an arrow.
(I) In vitro activity of wild-type and H163A mutant (Mut) DGAT2 for generation of acylceramide was determined.
(J) Microsomes from LacZ or DGAT2-expressing cells were incubated with PF 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.
Cell Metabolism  , DOI: ( /j.cmet )
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7. Figure 5
Modulation of Bioactive Ceramide Generation via ACSL5/DGAT2 Silencing and Overexpression
(A) Cellular growth was measured in cells transfected with Scr (control), ACSL1, or ACSL5 siRNAs.
(B) Cells were transfected with Scr (control) or ACSL5 siRNAs and treated with DMSO (control) or 5-FU (0.3 mM) and caspase 3/7 activity was measured. ACSL5 silencing was confirmed by western blotting (inset).
(C) Caspase 3/7 activity was measured in cells stably expressing LacZ or CerS1 after ACSL5 silencing.
(D) C16-ceramide levels were measured in cells transfected with Scr or DGAT2 siRNAs and treated with DMSO (control) or 5FU.
(E) Cells were transfected and treated as in (D), and caspase 3/7 activity was measured.
(F) Cells stably expressing control (vector) or HA-ACSL5 were treated with 5-FU and caspase 3/7 activity was measured. HA-ACSL5 expression was confirmed by western blotting (inset). Results are expressed as mean of three independent experiments with error bars representing ± SEM. ∗p < 0.05 and ∗∗p < 0.01.
(G) Caspase 3 activation and cleavage of PARP in response to 5-FU treatment was detected by western blotting in cells expressing control vector, ACSL5, or DGAT2.
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8. Figure 6 ACSL5, DGAT2, and CerS Interact on LD/ER interface
(A) Interaction of HA-tagged ACSL5 with V5-tagged DGAT2 was identified by anti-V5 IP and western blotting with anti-HA and anti-V5 antibodies.
(B) Cells expressing FLAG-CerS1 were transfected with control vector, HA-ACSL5, or V5-DGAT2 in combination with HA-ACSL5 and formation of ACSL5-DGAT2-CerS1 complex was determined by anti-FLAG IP and western blotting with anti-HA, anti-V5, and anti-FLAG antibodies. Results are representative of three independent experiments.
(C and D) Co-localization of HA-ACSL5 with V5-DGAT2 on ER (Calnexin) and LDs (BODIPY) was imaged using confocal microscopy in the absence (C) and presence of 0.5 mM oleate (D). Inset: magnified images of indicated areas.
(E and F) Co-localization of FLAG-CerS6 with V5-DGAT2 on ER (Calnexin) and LDs (BODIPY) was imaged in the absence (E) and presence of 0.5 mM oleate (F). Inset: magnified images of indicated areas.
(G) Localization of endogenous CerS6 and ACSL5 in LDs upon oleate treatment was detected using cellular fractionation followed by western blotting. LD, lipid droplet fractions.
(H) Total acylceramide content of LDs isolated from cells stably expressing vector control or DGAT2 was measured by LC-MS.
(I) Localization of CerS6 in LD fractions was determined by western blotting in cells stably transfected with control vector (left panel) or DGAT2 (right panel). LD, lipid droplet fractions. Results are representative of at least three independent experiments.
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9. Figure 7 Acylceramides Accumulate in Liver LDs of HFD-Fed Mice
(A) Liver sections of control diet or oleate HFD (4 weeks)-fed mice were stained with H&E or Oil Red O and visualized.
(B) Interaction of CerS6, ACSL5, and DGAT2 in liver lysates of control diet or oleate HFD (4 weeks)-fed mice (n = 3 for each group) was detected by IP with anti-CerS6 antibody followed by western blotting. SPT and ACER1 blotting was used as controls representing other ER resident proteins.
(C and D) Total ceramides (C) and total acylceramides (D) were measured in livers of mice that were fed a control diet or oleate-enriched HFD for 4 weeks using LC-MS.
(E and F) Total ceramide (E) and acylceramide (F) levels were measured in LDs from the livers of control diet or oleate-enriched HFD (4 weeks)-fed mice.
(G and H) Total acylceramide levels were measured in livers (G) and LDs from the livers (H) of mice that were fed a control diet or oleate-enriched HFD for 8 weeks with or without PF treatment.
Results are expressed as means (n = 4 for each group in 4 week study) (n = 3 for each group in 8 week study) with error bars representing ± SEM. ∗p < 0.05 and ∗∗p < 0.01.
Cell Metabolism  , DOI: ( /j.cmet )
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