Fig. 4: Long-chain ceramides are synthesised via the Cers2 de novo pathway. | Nature Communications

Fig. 4: Long-chain ceramides are synthesised via the Cers2 de novo pathway.

From: Long-chain ceramides are cell non-autonomous signals linking lipotoxicity to endoplasmic reticulum stress in skeletal muscle

Fig. 4: Long-chain ceramides are synthesised via the Cers2 de novo pathway.The alternative text for this image may have been generated using AI.

a The de novo synthesis pathway and salvage pathway of ceramide synthesis with de novo synthesis pathway inhibitors (red). Intracellular ceramides (Cer) in b C2C12 myotubes (control, 200 µM palmitate n = 4; 100 µM palmitate n = 3; one-way ANOVA; control vs 200 µM palmitate, Cer 40:1 P < 0.0001, Cer 42:1 P = 0.0026) (control = red, 100 µM palmitate = grey, 200 µM palmitate = blue) and c human skeletal muscle cells (HSkMCs) (control = red, 50 µM palmitate = grey, 100 µM palmitate = blue) (n = 4; one-way ANOVA; control vs 100 µM palmitate, Cer 40:1 P = 0.043; control vs 200 µM palmitate, Cer 40:1 P = 0.0003, Cer 42:1 P = 0.05; 100 µM palmitate vs 200 µM palmitate, Cer 40:1 P = 0.014) treated with palmitate. Long-chain ceramides in C2C12 myotubes co-treated with either d 10 µM myriocin (serine palmitoyl transferase inhibitor) (control = red, 10 µM myriocin = grey, 200 µM palmitate = blue, 200 µM palmitate + 10 µM myriocin = green) (n = 3; one-way ANOVA; control vs 10 µM myriocin, Cer 40:1 P < 0.0001, Cer 42:1 P < 0.0001; control vs 200 µM palmitate, Cer 40:1 P < 0.0001, Cer 42:1 P = 0.014; control vs 200 µM palmitate + 10 µM myriocin, Cer 40:1 P < 0.0001, Cer 42:1 P < 0.0001; 200 µM palmitate vs 10 µM myriocin, Cer 40:1 P < 0.0001, Cer 42:1 P < 0.0001; 200 µM palmitate vs 200 µM palmitate + 10 µM myriocin, Cer 40:1 P < 0.0001, Cer 42:1 P < 0.0001) or e 10 µM fumonisin B1 (FB1) (ceramide synthase (CerS) inhibitor) (control = red, 10 µM FB1 = grey, 200 µM palmitate = blue, 200 µM palmitate + 10 µM FB1 = green) (n = 4; one-way ANOVA; control vs 10 µM FB1, Cer 40:1 P < 0.0001, Cer 42:1 P < 0.0001; control vs 200 µM palmitate, Cer 40:1 P < 0.0001, Cer 42:1 P < 0.0001; 200 µM palmitate vs 10 µM FB1, Cer 40:1 P < 0.0001, Cer 42:1 P < 0.0001; 10 µM FB1 vs 200 µM palmitate + 10 µM FB1, Cer 40:1 P = 0.0001, Cer 42:1 P = 0.0005; 200 µM palmitate vs 200 µM palmitate + 10 µM FB1, Cer 40:1 P < 0.0001, Cer 42:1 P < 0.0001) and either 200 µM palmitate or bovine serum albumin (BSA). CerS2 expression in f. C2C12 myotubes (control = red; 100 µM palmitate = grey; 200 µM palmitate = blue) (control n = 3; 100 µM and 200 µM palmitate n = 4; one-way ANOVA; control vs 200 µM palmitate P = 0.044) and g. HSkMCs treated with palmitate or BSA (control = red; 50 µM palmitate = grey; 100 µM palmitate = blue) (n = 4; one-way ANOVA; control vs 100 µM palmitate P < 0.0001; 50 µM palmitate vs 100 µM palmitate P < 0.0001). h CERS2 expression in control HSkMCs (red) and HSkMCs treated with 100 µM palmitate (grey), scrambled control siRNA (con siRNA; white), siRNA against CERS2 (CERS2 siRNA; blue) or CERS2 siRNA and 100 µM palmitate (green) (n = 3; one-way ANOVA; control vs 100 µM palmitate P = 0.0016; control vs CERS2 siRNA P = 0.014; control vs CERS2 siRNA + 100 µM palmitate P = 0.011; 100 µM palmitate vs CERS2 siRNA P < 0.0001; 100 µM palmitate vs CERS2 siRNA + 100 µM palmitate P < 0.0001). i Cer 40:1 and Cer 42:1 normalised concentrations in control HSkMCs (red) and HSkMCs treated with 100 µM palmitate (grey), con siRNA (white), CERS2 siRNA (blue) or CERS2 siRNA and 100 µM palmitate (green) (n = 4; con siRNA n = 3; one-way ANOVA; control vs 100 µM palmitate, Cer 40:1 P < 0.0001, Cer 42:1 P = 0.001; control vs CERS2 siRNA Cer 40:1 P = 0.013, Cer 42:1 P = 0.01; 100 µM palmitate vs CERS2 siRNA Cer 40:1 P < 0.0001, Cer 42:1 P < 0.0001; 100 µM palmitate vs CERS2 siRNA + 100 µM palmitate Cer 40:1 P < 0.0001, Cer 42:1 P = 0.0001; CERS2 siRNA vs CERS2 siRNA + 100 µM palmitate Cer 40:1 P = 0.023). j Cer 40:1 and Cer 42:1 normalised concentrations in media conditioned on control HSkMCs (red) and HSkMCs treated with 100 µM palmitate (grey), con siRNA (white), CERS2 siRNA (blue) or CERS2 siRNA and 100 µM palmitate (green) (n = 4; one-way ANOVA; control vs 100 µM palmitate, Cer 40:1 P = 0.003, Cer 42:1 P = 0.0001; control vs CERS2 siRNA Cer 40:1 P = 0.0001, Cer 42:1 P = 0.0005; control vs CERS2 siRNA + 100 µM palmitate, Cer 40:1 P = 0.019, Cer 42:1 P = 0.011; 100 µM palmitate vs CERS2 siRNA Cer 40:1 P < 0.0001, Cer 42:1 P < 0.0001; 100 µM palmitate vs CERS2 siRNA + 100 µM palmitate Cer 40:1 P < 0.0001, Cer 42:1 P < 0.0001; CERS2 siRNA vs CERS2 siRNA + 100 µM palmitate Cer 40:1 P = 0.024). k UPR gene expression in HSkMCs receiving conditioned media from 100 μM palmitate or BSA-treated HSkMCs with and without CERS2 siRNA (control = red, 100 µM palmitate = grey, control + CERS2 siRNA = blue, 100 µM palmitate + CERS2 siRNA = green) (n = 3; one-way ANOVA; control vs 100 µM palmitate, ATF3 P = 0.045, HSPA5 P = 0.0043, EDEM1 P = 0.012; 100 µM palmitate vs CERS2 siRNA, ATF3 P = 0.003, HSPA5 P = 0.001, EDEM1 P = 0.0052; 100 µM palmitate vs CERS2 siRNA + 100 µM palmitate ATF3 P = 0.004, HSPA5 P = 0.0008, EDEM1 P = 0.004). *P ≤ 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are expressed as mean ± SEM with individual data points. Source data are provided as a Source Data file.

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