HIV-1 stock was generated using human embryonic kidney (HEK) 293T cells (ATCC) transfected with pYK-JRCSF plasmid containing full-length proviral DNA. Throughout the study, HBMEC were exposed to HIV-1 particles at the p24 level of 30 ng/ml as previously reported [22 (link)]. Treatment was terminated by removing cell culture media containing HIV-1, followed by washing the cells with PBS.
Aβ (1–40) and Aβ (1–40) HiLyte 647 were purchased from Anaspec (San Jose, CA) and dissolved in PBS. Freshly solubilized Aβ solutions without pre-aggregation were used for experiments as such a form of Aβ was demonstrated to induce proinflammatory reactions [33 (link)]. Aβ (1–40) HiLyte was dissolved first in a basic buffer (0.1 M NH4OH) and then diluted further in PBS as suggested by the manufacturer. Cells were treated with Aβ (1–40) or Aβ (1–40) HiLyte at the concentration of 100 nM in complete medium.
PAI039 (Tiplaxtinin, Catalog # PZ0295) was purchased from Millipore Sigma, Burlington, MA, USA). PAI039 is a potent and selective Serpine-1 inhibitor [34 (link)] and demonstrated efficacy in vivo in multiple models of acute arterial thrombosis. A 20 mM stock solution was prepared in DMSO. In a typical experiment, NPCs were cotreated with isolated EVs and/or 2 μM PAI039 for 24 h. Literature indicates that 1 μM PAI-039 can effectively inhibit Serpine-1 activity in vitro [35 (link)]. PAI039 exerts its activity by binding close to the vitronectin binding site [36 (link)].
Aβ (1–40) and Aβ (1–40) HiLyte 647 were purchased from Anaspec (San Jose, CA) and dissolved in PBS. Freshly solubilized Aβ solutions without pre-aggregation were used for experiments as such a form of Aβ was demonstrated to induce proinflammatory reactions [33 (link)]. Aβ (1–40) HiLyte was dissolved first in a basic buffer (0.1 M NH4OH) and then diluted further in PBS as suggested by the manufacturer. Cells were treated with Aβ (1–40) or Aβ (1–40) HiLyte at the concentration of 100 nM in complete medium.
PAI039 (Tiplaxtinin, Catalog # PZ0295) was purchased from Millipore Sigma, Burlington, MA, USA). PAI039 is a potent and selective Serpine-1 inhibitor [34 (link)] and demonstrated efficacy in vivo in multiple models of acute arterial thrombosis. A 20 mM stock solution was prepared in DMSO. In a typical experiment, NPCs were cotreated with isolated EVs and/or 2 μM PAI039 for 24 h. Literature indicates that 1 μM PAI-039 can effectively inhibit Serpine-1 activity in vitro [35 (link)]. PAI039 exerts its activity by binding close to the vitronectin binding site [36 (link)].
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