Figure 1.

Extracellular communication shown through functional transfer of Cre activity in vitro

(A) Top: schematic representation of the lentiviral construct expressing nuclear localization signal (NLS) CRE (1,026 bp) under control of PGK promoter, and H2B firefly luciferase (Fluc) (1,650 bp) under control of UBC promoter. Cre and Fluc genes contain a NLS and H2B, respectively, at the N terminus that shuttles the proteins to the nucleus. Bottom: representative immunofluorescent image from confocal microscopy of HEK293T cells stably expressing Cre protein (red) mainly in the nucleus (blue). Actin filaments in cytoplasm were stained with phalloidin (white). Scale bar, 10 μm. (B) Schematic representation of FLExNanoluc switch used to generate a sensitive Cre reporter system. The FLExNanoluc in the OFF-state does not allow Nanoluciferase (Nanoluc) expression, because the gene is backward in the construct. Upon Cre activation, the Nanoluc gene flips and becomes in frame with the EF1α promoter in the ON-state. The resulting Nanoluc expression generates detectable bioluminescence in both cells and medium. (C) Co-culture of HEK293T cells stably expressing Cre (red) and HEK293T cells stably expressing FLExNanoluc and GFP (green) for 72 h. Scale bar, 20 μm. (D) Bioluminescence evaluation of Nanoluc secreted in medium. Nanoluc signal in the cell medium detected after 24 and 72 h of co-culture. Cells were cultured in three FLExNanoluc:Cre ratios (1:1, 1:3, and 3:1). The white bars represent a control condition in which FLExNanoluc reporter cells were co-cultured with WT HEK293T cells (no expression of Cre). Cre activity is represented by a bioluminescence signal relative to control (n = 6). Data are presented as mean ± SEM and compared by unpaired t test, ∗∗∗∗p < 0.0001. (E) Transwell system (1 μm pore inserts) with Cre cells seeded on the apical side of the upper chamber and previously transfected with CMV-STEAP3-SDC4-NadB plasmid to boost small EV production and FLExNanoluc reporter cells seeded in the lower chamber, with the latter showing recombination mediated by EVs. (F) Cre activity in boosted condition relative to non-boosted condition is represented by Nanoluc bioluminescence (RLU) in FLEx cells (n = 3). Data are presented as mean ± SEM and compared by unpaired t test, ∗∗p < 0.01. (G) Evaluation of gDNA recombination by RT-PCR showing Ct values of non-recombined DNA (FLExOFF) and recombined DNA (FLExON) (n = 3/4). FLEx condition (white bar) was used to establish a baseline condition corresponding to no recombination. Data represented as Ct values obtained in each sample condition. Data are presented as mean ± SEM and compared by one-way ANOVA followed by Tukey’s multiple comparison test (F = 19.72, F = 6.956); ∗p < 0.05, ∗∗p < 0.01.