Fig. 4.

PTTG1IP-EV-mediated delivery of Cre recombinase to reporter cells.

HEK293T cells were transfected with various PTTG1IP constructs and the capacity to transfer Cre protein was tested in reporter cells. (A) Schematic of tested Cre fusion constructs. SC is a self-cleaving endopeptidase from Shewanella oneidensis. The intein is a self-cleaving sequence from Mycobacterium tuberculosis. (B) Timeline of EV-transfer experiment to Cre reporter cells. 24 h after donor cell transfection with Cre constructs and VSVG, media is replaced with fresh Opti-MEM and EVs are isolated 48 h later by SEC. Isolated EVs are then transferred to recipient cells in 96-well plates (5 × 10⁹ EV/ml). 24 h later media is replaced with DMEM 10% FBS and after 24 h cells are analysed for GFP expression by flow cytometry. (C) Schematic of the reporter system in the Cre reporter cells. RFP is constitutively expressed by reporter cells while GFP is only expressed when the floxed stop cassette is excised by Cre. (D) GFP positive cells quantified by flow cytometry 48 h after EV-transfer to T47D cells. (E) GFP positive cells quantified by flow cytometry 48 h after EV-transfer to HeLa cells. (F) Western blot of Cre-fusion constructs in HEK293T EVs and cells. Both cell lysates and isolated EVs were blotted using antibodies against MYC. Cell lysates and isolated EVs were blotted for ACTB and ALIX, respectively. Statistical significance was tested by one-way ANOVA and Bonferroni post hoc test. Data are presented as mean +SEM, n = 3 replicates, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.