C3505

H1299- and HCC827-PD-L1-overexpressed cells were exposed to medium containing 1% FBS for 12 h and then treated with the following inhibitors separately: the autophagy inhibitor 3-methyladenine (1 mM, Selleck, S2767), the protein transport inhibitor brefeldin A (10 ng/ml, APExBIO, B1400), and the exosome secretion inhibitor 5-(N,N-dimethyl)-amiloride DMA (50 nM, APExBIO, C3505). The supernatant was collected from the cultured medium 24 h later. Gas6 level was measured with a human Gas6 ELISA kit (SEA204Hu, USCN Life Sciences, Wuhan, China).

The levels of human (SEC642Hu) and mouse serum MSN (SEC642Mu) and PCT (SEA689Mu) were quantified by ELISA using specific kits (USCN Life Sciences, Wuhan, China), according to the manufacturer's instructions. To explore the secretion mechanisms of MSN in HMECs after LPS stimulation, three inhibitors of the main pathways of protein secretion were employed in the experiment. The dosage of inhibitors is as follows: the autophagy inhibitor 3-methyladenine (3-MA, 1 mM, Selleck, S2767), the exosome secretion inhibitor 5-(N,N-dimethyl)-amiloride DMA (DMA, 50 nM, APExBIO, C3505), and the protein transport inhibitor brefeldin A (BFA, 10 ng/ml, APExBIO, B1400). The supernatants were collected from the cultured medium 24 h later. To explore the role of MSN in the process of excessive inflammation caused by sepsis, HMECs were, respectively, transfected with siNC and siMSN RNA for 48 h, then exposed to LPS for 24 h, and the supernatants were collected to measure IL-1β, IL-18, IL-6, and TNF-α levels (E-EL-H0149c, IL-18 E-EL-H0253c, E-EL-H0102c, E-EL-H0109c, Elabscience Biotechnology, Wuhan, China).