Fig. 2. HSPC EVs remodel the EC secretome through NF-κB activation.

(A) Transcriptional analysis of murine BM ECs by RT² Profiler PCR Array. ECs were cultured in isolation or in noncontact 1.0-μm transwell dishes with murine HSPCs for comparative analysis. (B) Relative gene expression of target chemokines following transfer of HSPC or plasma-derived EVs (10³ to 10⁴ EV per cell). (C) Known receptor-ligand binding partners of CCR1 to CCR3. (D) Relative gene expression of CCR2 ligand (CCR2L) receptors on HSPCs after 1 to 2 days in transwell culture with ECs. Dotted line denotes isolated HSPCs. (E) Relative surface expression of CCR1 to CCR3 on HSPCs after 2 days in transwell EC culture. Dotted line denotes isolated HSPCs. (F) Surface expression of CCR2 and CXCR4 on HSPC subpopulations. (G) Representative immunoblots and (H) quantitative analysis demonstrating a dose-dependent activation of canonical NF-κB signaling in ECs after HSPC EV uptake. (I) Relative gene expression of CCR2L in ECs after HSPC EV transfer and treatment with the IκB kinase inhibitor, ACHP. (J) Relative gene expression of CCR2L and Cxcl12 after HSPC EV uptake into WT ECs (dotted line) or cells transduced with an IκBα dominant-negative (DN) mutant. Iso, isolated; TW, transwell. *P < 0.05, **P < 0.01, and ***P < 0.001.