Fig. 2: Long-chain ceramides activate the unfolded protein response in myotubes. | Nature Communications

Fig. 2: Long-chain ceramides activate the unfolded protein response in myotubes.

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

Fig. 2

a Diacyglycerols (DG), lysophosphocholines (LPC) and ceramides (Cer) are enriched in conditioned media collected from C2C12 myotubes treated with vehicle control (red) 100 μM (grey) and 200 μM (blue) palmitate (n = 4; one-way ANOVA; control vs 200 µM palmitate, DG 32:0 P < 0.0001, DG 34:0 P = 0.002, DG 34:1 P = 0.0002, DG 36:0 P = 0.0068, DG 36:1 P = 0.0025, LPC 16:0 P < 0.0001, LPC 16:1 P = 0.0001, LPC 18:0 P = 0.0029, Cer 34:1 P = 0.0012, Cer 40:1 P < 0.0001, Cer 42:1 P = 0.004; control vs 100 µM palmitate, Cer 40:1 P = 0.043; 100 µM palmitate vs 200 µM palmitate, DG 32:0 P < 0.0001, DG 34:0 P = 0.013, DG 34:1 P = 0.0006, DG 36:0 P = 0.023, DG 36:1 P = 0.0043, LPC 16:0 P = 0.0008, LPC 16:1 P = 0.0036, LPC 18:0 P = 0.0043, Cer 34:1 P = 0.002, Cer 40:1 P = 0.001, Cer 42:1 P = 0.02). UPR gene expression in C2C12 myotubes treated with vehicle control (red) and lipids: b 5 μM (grey) and 10 μM (blue) DG 32:0 (n = 4), c 10 μM (grey) and 100 μM (blue) LPC 16:0 (control n = 3; 10 μM and 100 μM LPC 16:0 n = 4), d 5 μM (grey) and 50 μM (blue) LPC 18:0 (n = 4), e 5 μM (grey) and 10 μM (blue) Cer 40:1 (n = 4; one-way ANOVA; control vs 10 µM Cer 40:1, Atf4 P = 0.023, Hspa5 P = 0.05) and f 5 μM (grey) and 10 μM (blue) Cer 42:1 (n = 4; one-way ANOVA; control vs 10 µM Cer 42:1, Atf4 P = 0.02, Hspa5 P = 0.036). g UPR gene expression in C2C12 myotubes treated with vehicle control (red) and a combination of 10 μM Cer 40:1 and 10 μM Cer 42:1 (grey) (n = 3; two-tailed Student’s t test, Atf4 P = 0.007, Hspa5 P = 0.017, Edem1 P = 0.05). h Cer 40:1 and Cer 42:1 concentrations in conditioned media from human skeletal muscle cells (HSkMCs) treated with 50 μM (grey) and 100 μM (blue) palmitate or vehicle control (red) (n = 4; one-way ANOVA; control vs 50 µM palmitate, Cer 40:1 P = 0.001, Cer 42:1 P = 0.025; control vs 100 µM palmitate Cer 40:1 P < 0.0001, Cer 42:1 P = 0.01; 50 µM palmitate vs 100 µM palmitate Cer 40:1 P = 0.014). i UPR gene expression in HSkMCs treated with vehicle control (red) or a combination of 10 μM Cer 40:1 and 10 μM Cer 42:1 (grey) (n = 4; two-tailed Student’s t test; ATF3 P = 0.001, ATF4 P = 0.016, EDEM1 P = 0.014). j Western blot for C/EBP Homologous Protein (CHOP), X-box binding protein 1 active splice variant (XBP-1s) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression control from HSkMCs treated with 10 µM ceramides 40:1 and 42:1 and a combination of both ceramides. k Normalised Western blot densitometry for CHOP and XBP-1s from HSkMCs treated with vehicle control (red) 10 µM ceramides 40:1 (grey) and 42:1 (blue) and a combination of both ceramides (green) (n = 3; one-way ANOVA; Control vs 10 µM Cer 40:1 and Cer 42:1, XBP-1s P < 0.0001, CHOP P = 0.0018; Control vs 10 µM Cer 42:1, CHOP P = 0.0005). l Confocal microscopy of HSkMCs expressing endoplasmic reticulum-targeted red fluorescent protein (ER-RFP; red) and treated with vehicle control, 10 µM Cer 40:1 or 10 µM Cer 42:1, or a combination of both 10 µM Cer 40:1 and Cer 42:1. Nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar = 20 µm. m The normalised ER-RFP fluorescence density in HSkMCs expressing ER-RFP and treated with vehicle control (red), 10 µM Cer 40:1 (grey) or 10 µM Cer 42:1 (blue) or a combination of both 10 μM Cer 40:1 and Cer 42:1 (green) (n = 3; one-way ANOVA; control vs 10 µM Cer 40:1 and Cer 42:1, P = 0.0005). *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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