Figure 1.

Long Interspersed Element-1 (LINE-1) induction by benzo[a]pyrene and its impact on extracellular vesicle (EV) LINE-1 content, number, and size. (A) LINE-1 element domain structure. LINE-1 is a 6 kb repetitive DNA element that comprises 5′ and 3′ untranslated regions (UTRs) and two proteins, ORF1p and ORF2p. ORF1p is a 40 kDa nucleic acid binding protein that forms multimers. ORF2p is 150 kDa and contains both endonuclease (EN) and reverse transcriptase (RT) domains. ORF1p trimers and ORF2p exhibit strong affinity for the LINE-1 mRNA, and together form a ribonucleoprotein (RNP) containing both the template and enzymatic machinery required for target-site primed reverse transcription. (B) ORF1p induction in H460 cells by benzo[a]pyrene (BaP). To examine LINE-1 content in EVs, we used a LINE-1 inducer, the carcinogen BaP. (C) EV preparation. EVs were collected from cells challenged with 1 μM BaP for 48 h. The size and morphology of the preparation was assessed using Nanosight Nanoparticle Tracking Analysis (NTA) (top) and electron microscopy (bottom). (D) H460 EV LINE-1 content. BaP exposure increased EV LINE-1 mRNA and ORF1p content in BaP treatments compared to DMSO controls (as measured by One-Step RT-qPCR and ELISA; * p < 0.05; Welch’s t-test) but did not significantly alter the total abundance of EVs (p > 0.05; t-test), as shown in (E). NTA of BaP and DMSO treatments (F). (G) BaP-induced size differences. To examine subtle changes in EV size, total EV numbers were normalized to 5000 particles per reading, and each EV size bin was compared between treatments using a two-way ANOVA. Yellow bars indicated EV sizes with significant differences. N = 3–5. (H) Test gradient (BaP vs. DMSO control). For subsequent density gradient EV studies in this cell line, we performed a test gradient and determined that BaP exposure (red) was necessary to raise LINE-1 expression to detectable levels. * Difference between DMSO and BaP (p < 0.05; two-way ANOVA).