Fig. 7: UPR-inducing ceramides are secreted via extracellular vesicles. | Nature Communications

Fig. 7: UPR-inducing ceramides are secreted via extracellular vesicles.

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

Fig. 7

a Extracellular vesicle (EV) population images in conditioned media from C2C12 myotubes treated with either 200 μM palmitate (grey) or bovine serum albumin (BSA) control (red). b The small EV population secreted by C2C12 myotubes treated with either 200 μM palmitate (grey) or BSA vehicle (red) (n = 4; two-tailed Student’s t test; P = 0.02). c The large EV population secreted by C2C12 myotubes treated with either 200 μM palmitate (grey) or the BSA vehicle (red) (n = 4; two-tailed Student’s t test; P < 0.0001). Activating transcription factor 3 (Atf3), activating transcription factor 4 (Atf4), Heat Shock Protein Family A (Hsp70) Member 5 (Hspa5) and ER Degradation Enhancing Alpha-Mannosidase Like Protein 1 (Edem1) UPR gene expression in myotubes treated with d. small EVs (n = 4; two-tailed Student’s t test; Atf3 P = 0.05, Atf4 P = 0.02; Edem1 P = 0.05) and e. large EVs (n = 3; two-tailed Student’s t test; Atf3 P = 0.03, Atf4 P = 0.007; Hspa5 = 0.01, Edem1 P = 0.02) isolated from the media of palmitate (grey) and BSA-treated (red) myotubes. f UPR gene expression in myotubes treated with conditioned EV-depleted media from 200 μM palmitate-treated (grey) and control (red) myotubes (n = 3; two-tailed Student’s t test; Atf3 P = 0.002, Atf4 P = 0.04, Edem1 P = 0.05). Ceramide (Cer) 40:1 and Cer 42:1 concentration in g. small EVs (n = 4; two-tailed Student’s t test; Cer 40:1 P = 0.0004, Cer 42:1 P = 0.0006) and h. large EVs (n = 4; two-tailed Student’s t test; Cer 40:1 P < 0.0001, Cer 42:1 P = 0.001) from the media of C2C12 myotubes treated with either 200 μM palmitate (grey) or BSA control (red). i Cer 40:1 and Cer 42:1 concentration in EV-containing and EV-depleted media from C2C12 myotubes treated with either 200 μM palmitate or BSA control (control = red, control EV-depleted = grey, palmitate = blue, palmitate EV-depleted = green) (n = 4; one-way ANOVA; control vs 200 µM palmitate, Cer 40:1 P = 0.0007, Cer 42:1 P = 0.012; control vs control EV-depleted, Cer 40:1 P = 0.0011, Cer 42:1 P < 0.0001; control vs 200 µM palmitate EV-depleted, Cer 40:1 P = 0.0016, Cer 42:1 P < 0.0001; 200 µM palmitate vs control EV-depleted, Cer 40:1 P < 0.0001, Cer 42:1 P < 0.0001; 200 µM palmitate vs 200 µM palmitate EV-depleted, Cer 40:1 P < 0.0001, Cer 42:1 P < 0.0001). *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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