FIGURE 1.

IFN‐γ and Ca²⁺ promote sEV PD‐L1 secretion in H1299 cells. (a) A representative transmission electron microscopy (TEM) image of a purified sEV from H1299 cells. Scale bar, 100 nm. (b) sEVs were isolated by sucrose gradient density ultracentrifugation, followed by the immunoblotting for PD‐L1 and sEVs markers CD63 and HRS. (c) Nanoparticle tracking analysis (NTA) was conducted on H1299 cells to determine the size distribution of sEVs isolated by ultracentrifugation. Traces represent mean values in units of million particles per ml from 3 experiments. (d) Immunoblots for PD‐L1 and sEVs markers ALIX, CD63 and CD9 in whole‐cell lysates and sEVs from H1299 cells treated with or without IFN‐γ (10 ng/ml). (e) Immunoblotting analysis of PD‐L1, EVs markers and organelle makers performed on whole‐cell lysates (WC), m/lEVs and sEVs from H1299 cells. (f) Effect of IFN‐γ pretreatment on thapsigargin‐(Tg 2 μM) and ATP‐(100 μM) mediated Ca²⁺ dynamics. Values of fluorescent intensity represent mean ± SEM, which is normalized to the basic fluorescence intensity. All data is derived from more than 4 wells in 3 individual experiments. (g) Schematic illustration of PD‐L1_pHluorin plasmid (upper) and proposed sEVs releasing model (below) through visualization of PD‐L1_pHluorin. sEV PD‐L1_pHluorins are quenched while resident in the acidic lumen of the multivesicular body (MVB). Immediately after sEVs secretion, the pHluorin is exposed to the neutral extracellular medium, causing a sudden increase in fluorescence intensity. (h) Total projection of secretion events over a 180 s was recorded from a cell before (top) and after (bottom) stimulation with Tg (2 μM). (i) Counts of fusion events within a time window of 180 s in individual H1299 cells (n = 11) transfected with PD‐L1_pHluorin. Two points connected by a line represent the fusion events numbers from the same cell but under different conditions (before Tg vs. after Tg). For all of Figure 1, data are representative of at least three independent experiments. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.