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. 2022 Dec 14;13(4):1568–1587. doi: 10.1016/j.apsb.2022.12.008

Figure 2.

Figure 2

METRNL secretion by endothelial cells involves the classical ER–Golgi pathway. (A) Domain structure of human METRNL with the N-terminal signal peptide (SP) (up). METRNL mRNA expression (left) in HUVECs with adeno-associated virus (AAV)-mediated METRNL overexpression and METRNL concentrations (right) in the serum-free culture medium during 24 h (n = 4–6). (B) METRNL mRNA expression (left) in HUVECs with lentivirus-mediated METRNL knockdown and METRNL concentrations (right) in the serum-free culture medium during 24 h (n = 4–6). (C) Domain structure of human METRNL without SP region (METRNL-ΔSP, up). METRNL mRNA expression (left) in HUVECs with AAV-mediated overexpression of METRNL without SP domain and METRNL concentrations (right) in the serum-free culture medium during 24 h (n = 3–4). (D) METRNL secretion from primary cultured HUVECs inhibited by both eeyarestatin 1 (5 μg/mL) and brefeldin A (5 μg/mL) (n = 3). NT, no treatment as blank control. (E) Recovery of METRNL release from HUVECs after brefeldin A removal. Brefeldin A (5 μg/mL) or a solvent control (DMSO) was added to the serum-free culture medium 3 h before medium collection for METRNL measurement. Then the cells were washed for three times and incubated in normal culture medium without drugs for 16 h followed by another 3 h of serum-free medium incubation and METRNL detection of the culture supernatant (n = 3). NT, no treatment as blank control. Data are mean ± SEM. ∗∗P < 0.01, ∗∗∗P < 0.001 by two-tailed Student's t test in (A, B and C). ∗∗∗P < 0.001 vs. Vehicle; ###P < 0.001 vs. NT by one-way ANOVA followed by Dunnett's t test in (D and E).