1. Very Long Chain Fatty Acids Are Functionally Involved in Necroptosis
Laura R. Parisi, Nasi Li, G. Ekin Atilla-Gokcumen 
Cell Chemical Biology 
Volume 24, Issue 12, Pages e8 (December 2017)
DOI: /j.chembiol
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2. Cell Chemical Biology 2017 24, 1445-1454. e8DOI: (10. 1016/j. chembiol
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3. Figure 1 Characterization of Necroptotic Cell Death
(A) Viability of control, necroptotic, and Nec-1s-protected cells. Necroptosis results in a drastic reduction in cell viability and is completely prevented by RIPK1 inhibitor Nec-1s. Error bars represent 1 SD; n = 5.
(B) Western blots of control and necroptotic cell lysates. Cleaved PARP (clv. PARP) and phosphorylated MLKL (p-MLKL) are markers for apoptosis and necroptosis, respectively.
(C) Fluorescence microscopy images of control and necroptotic cells. DAPI (green in overlay) is used to visualize all cell nuclei; propidium iodide (PI, red in overlay) uptake indicates plasma membrane permeabilization, which occurs in necroptotic but not in control cells. Scale bar, 50 μm.
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4. Figure 2
Targeted Analysis of Representative Lipids from Major Lipid Classes in Control and Necroptotic Cells
Ceramides and specific fatty acids constitute the most profound changes in the lipidome during necroptosis. The heatmap shows the relative abundances of each species, calculated by dividing the abundance of a lipid by the average abundance of that lipid in all (control and necroptotic) samples. FA, fatty acid; (L)PC, (lyso)phosphatidylcholine; (L)PA, (lyso)phosphatidic acid; (L)PI, (lyso)phosphatidylinositol; (L)PE, (lyso)phosphatidylethanolamine; PS, phosphatidylserine; Cer, ceramide; DiHCer, dihydroceramide; SM, sphingomyelin; HexCer, hexosylceramide; DiHexCer, dihexosylceramide; MAG, monoacylglycerol; DAG, diacylglycerol; TAG, triacylglycerol. See also Tables S1 and S2 for related information.
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5. Figure 3
Accumulation of Ceramides and Very Long Chain Fatty Acids is RIPK1 Dependent
(A) Targeted analysis of fatty acids in necroptotic and Nec-1s-protected cells. Necroptosis fold changes (gray dots) represent Abundancenecroptotic/Abundancecontrol. Nec-1s-protected fold changes (black triangles) represent AbundanceNec-1s-protected/Abundancecontrol. Nec-1s prevents accumulation of very long chain fatty acids, restoring them to near basal levels (fold change ≈ 1). Dashed line indicates fold change = 1. For C23-, C24-, and C26-fatty acids, a numeric value for fold change could not be calculated because it was not detected in control or Nec-1s-protected cells. Instead, the increase during necroptosis is represented as “High.”
(B) Targeted analysis of ceramides in necroptotic and Nec-1s-protected cells. Necroptosis fold changes (gray dots) represent Abundancenecroptotic/Abundancecontrol. Nec-1s-protected fold changes (black triangles) represent AbundanceNec-1s-protected/Abundancecontrol. Nec-1s prevents ceramide accumulation, restoring ceramides to basal levels (fold change ≈ 1). Dashed line indicates fold change = 1. For C18- to C20-ceramides, a numeric value for fold increase during necroptosis could not be calculated due to the low abundance of these species in control and Nec-1s-protected cells. Instead, the increases during necroptosis are represented as “High.”
Error bars represent ±1 SD; n = 4. *p < 0.05, **p < 0.01; ***p <
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6. Figure 4
Genes Involved in Fatty Acid Biosynthesis, Elongation, and De Novo Ceramide Biosynthesis Are Upregulated during Necroptosis
(A) Simplified scheme of fatty acid and sphingolipid biosynthesis. Shown in bold are the transcripts and lipids that increase during necroptosis. ACLY, ATP citrate lyase; ACC, acetyl-CoA carboxylase; FASN, fatty acid synthase; ELOVLs, elongation of very long chain fatty acid proteins; SCD, stearoyl-CoA desaturase; FADs, fatty acid desaturases; CERSs, ceramide synthases; nSMase, neutral sphingomyelinase; aSMase, acid sphingomyelinase.
(B) Fold change in expression of ACLY, ACACA, FASN, elongases ELOVL1, 3, 5, 6, and 7, and ceramide synthases CERS1–6. Fold change is calculated as the ratio of relative expression of each gene compared with HPRT1 in necroptotic and control cells. Increased expression of CERSs and FASN/ELOVL are likely responsible for the accumulation of ceramides and very long chain fatty acids during necroptosis. Gene expression was measured by droplet digital PCR; expression of CERS4 and ELOVL5 was not detected. Dashed line indicates fold change = 1. See STAR Methods for description of error bars; n = 3–6. *p < 0.05, **p < The sequences of primers are provided in Table S3.
See also Figure S5 for related information.
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7. Figure 5
Inactivation of FASN Prevents VLCFA Accumulation, Cell Death, and Membrane Permeabilization due to Necroptosis
(A) Structures of inhibitors of fatty acid (FA) biosynthesis/uptake. FASN, fatty acid synthase; ACC, acetyl-CoA carboxylase.
(B) Cell viability in the absence (No Inh) or presence of fatty acid biosynthesis/uptake inhibitors in control and necroptotic cells. Cerulenin, a fatty acid synthase inhibitor, results in significant protection from necroptotic cell death, suggesting a functional role of very long chain fatty acids in necroptosis. We note that the difference in necroptotic cell viability with no inhibitor compared with Figure 1A is due to shorter (3 hr) TNF-α treatment and increased cell number following 24 hr of pre-treatment (see STAR Methods). Error bars represent 1 SD; n ≥ 5. **p < 0.01, ***p < relative to cell viability during necroptosis with no inhibitor, which is indicated by the dashed line.
(C) Necroptotic activity of shFASN cells. We knocked down FASN in HT-29 cells using a lentiviral shRNA vector (see Figures S6B and S6C). Compared with a non-target (red fluorescent protein, shRFP) control, cell viability during necroptosis was increased up to 3-fold in FASN knockdown cells (shFASN). We note the difference in cell viability in shRFP cells compared with control cells in (B), which is in part due to effects of lentiviral transduction, as well as difference in cell number (see STAR Methods). Error bars represent 1 SD; n = 5. ***p < relative to shRFP cell viability during necroptosis, which is indicated by the dashed line.
(D) Targeted analysis of fatty acids in necroptotic and cerulenin-protected cells compared with control cells. Necroptosis fold changes (black bars) represent Abundancenecroptotic/Abundancecontrol. Cerulenin-protection fold changes (gray bars) represent Abundancecerulenin-protected/Abundancecontrol. Cerulenin significantly prevents accumulation of C24:0 and C26:0 fatty acids due to necroptosis, suggesting a functional link between these specific fatty acids and the execution of necroptotic cell death. The other very long chain fatty acid that accumulated during necroptosis, C23-fatty acid, was not detected in cerulenin-protected or necroptotic cells, which we attribute to the shorter TNF-α treatment. Error bars represent 1 SD; n = 3. *represents p < 0.05; ** represents p < 0.01.
(E) Propidium iodide uptake. Cerulenin prevents plasma membrane permeabilization due to necroptosis as indicated by reduced fluorescence due to propidium iodide uptake. Error bars represent 1 SD; n = 5.
See also Figure S6 and Table S4 for related information.
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