Fig. 2: aPC-triggered EV generation from human lung cancer cells involves intracellular activation of the RhoA-ROCKII-JNK1/2-MLC2 pathway.

A A549 cells were serum starved for 1 h followed by the pre-treatment of Rhosin (50 µM) or Y27632 (10 µM) or JNK I (SP600125; 20 µM) for another 1 h. Cells were then challenged with a CV or aPC (25 nM) for 16 h. EVs in the supernatant were quantified by NTA Nano-Sight. B Cells were starved for 1 h followed by treatment with a CV or aPC (25 nM). Cells were lysed after 10 min, 1 h, and 2 h of treatment to analyze the level of RhoA-GTP level and phosphorylation of JNK1/2, and MLC2, respectively by western blotting. RhoA activation was measured by analyzing the GTP-bound RhoA using the RhoA activation assay kit. C Band intensities were quantified by densitometric analysis. D Cells were pre-treated with Rhosin for 1 h followed by CV or aPC challenge. RhoA activation and phosphorylation of JNK1/2 and MLC2 were measured. E Band intensities were quantified after performing the densitometric analysis. F Cells were serum-starved for 1 h followed by pre-treatment with a CV or Y27632 for another 1 h. After the indicated time, cells were challenged with a CV or aPC and RhoA, JNK1/2, and MLC2 activation was analyzed after 10 min, 1 h, and 2 h, respectively. G Band intensities were quantified after densitometric analysis. H Serum-starved cells were treated with a CV or JNK I for 1 h, followed by the exposure of a CV or aPC. After 10 min and 2 h, RhoA activation and MLC2 phosphorylation were analyzed and I accordingly densitometric analysis was performed to quantify band intensities. J Schematic representation demonstrates that aPC binding to EPCR triggers PAR1 signaling to induce RhoA activation. The RhoA signaling promotes ROCKII activation which further promotes JNK1/2 phosphorylation. Phospho-JNK1/2 further promotes MLC2 phosphorylation, which triggers the release of EVs. ***P < 0.001; ****P < 0.0001; ns not statistically significantly different.