Figure 5.

Functional analysis of Nef^mut/43M2 engineered exosomes. (A) Viability of SiHa cells after challenge with Nef^mut/43M2-uploading exosomes. RTCA proliferation assay in both HPV18-positive HeLa and HPV16-positive SiHa cells after transfection with either Nef^mut/GO or Nef^mut/43M2 expressing vectors. Results shown are representative of five independent experiments. (B) RTCA proliferation assay in both HeLa and SiHa cells challenged with exosomes uploading either Nef^mut/GO or Nef^mut/43M2. As an additional control, SiHa cells were challenged with equivalent amounts of exosomes isolated from the supernatants of mock-transfected cells. Arrows indicate the time of exosome treatment, i.e., 24 hrs after seeding. Results are expressed as normalized cell index, i.e., the ratio between cell index at a given time and cell index at the time of exosome treatment, and are representative of two independent experiments. (C) Viability of SiHa cells co-cultured in transwell chambers with HEK293T cells producing Nef^mut/43M2 engineered EVs. HEK293T cells were transfected with vectors expressing either Nef^mut/43M2, Nef^mut/GO, or empty vector. After 24 hrs, transfected cells were seeded in the upper transwell chamber while either SiHa or Hela cells were plated in the lower one. Four days later, cell viability was assessed by MTS-based cell proliferation assay. At this time, cells from each well were detached, resuspended in 0.4 mL of complete medium, and four microwells of a 96-well plate were seeded with 100 µL of each cell culture. Shown are mean OD values + standard errors from the results of six independent experiments performed in quadruplicates. *p: 0.0094; **p: 0.0274 by two-tailed Mann–Whitney U-Test. (D) Cell viability as assessed by MTS-based cell proliferation assay 4 days after setting the co-culture of SiHa cells with HEK293T cells previously transfected with the indicated vectors, and carried out in the presence or absence of 1 µM GW4869 and spiroepoxide. SiHa cell cultures were processed for the MTS assay as described for panel A. Shown are mean OD values + standard errors from the results of four independent experiments conducted in quadruplicates. *p: 0.0396 by two-tailed Student T Test. Differences in cell viability between SiHa cells co-cultivated with donor cells transfected with the two different vectors in the presence of the inhibitors were not statistically significant (p: 0.5471 by two-tailed Student T Test).