Figure 2

(A) Schematic of peroxisome-mediated lipid metabolism. VLCFA, very-long-chain fatty acids. (B) Pie charts showing lipid composition (relative abundance of each lipid family in percentage total) in D4M.3a Cas9-Ctrl, Pex3^+/– clone 6D, and Pex3^+/– clone 9G cells. Concentrations of each lipid family (normalized to mg DNA) are indicated (n = 3). (C) Volcano plots comparing abundance of lipid species in clone 6D versus Cas9-Ctrl (left) and clone 9G versus Cas9-Ctrl (right). Yellow and blue shades highlight respective increased (≥1.5-fold) and decreased (≤1.5-fold) lipid species in Pex3^+/– cells relative to Cas9-Ctrl D4M.3a cells. (D) Venn diagrams showing lipid species that were significantly increased (top) or decreased (bottom) in D4M.3a clone 6D and clone 9G cells, compared with Cas9-Ctrl cells. (E) Top: Heatmap showing lipid species that were commonly altered in D4M.3a Pex3^+/– (6D and 9G) cells compared with Cas9-Ctrl cells. Bottom: Number of lipid species, categorized by family, enriched in D4M.3a Cas9-Ctrl or Pex3^+/– (6D and 9G) cells. PC, phosphatidylcholine; PE, phosphatidylethanolamine; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; TG, triglyceride; PE-O, 1-alkyl,2-acylphosphatidylethanolamine; PC-O, 1-alkyl,2-acylphosphatidylcholine; PC-P, 1-alkenyl,2-acylphosphatidylcholine; PE-P, 1-alkenyl,2-acylphosphatidylethanolamine; DG, diacylglyceride; CE, cholesterol ester. (F) Concentrations of ceramides (left) and hexosylceramides (HexCer, right) detected in D4M.3a Cas9-Ctrl, 6D, and 9G cells (n = 3). Two-way ANOVA. (G–I) Percentage apoptosis (PI⁺/Annexin V⁺, PI^–/Annexin V⁺) detected in DMSO- or vemu-treated (G and H) D4M.3a Cas9-Ctrl (left) or Pex3^+/– clone 9G (right) or (I) A375M cells. Cells were pretreated with (G and I) C2-ceramide (C2-Cer) at escalated doses or with (H and I) C2-Cer (10 μM) and vorinostat (Vor, 1 μM) 24 hours prior to vemu treatment (n = 4 for H, n = 3 for G, I). Two-way ANOVA. All data represent mean ± SD.