Figure 5.

Assessment of the impact of PE N/MPL chronic exposure on vaginal keratinocytes viability, morphology, proliferation, inflammation, epigenetic regulation, and transformation potential. (a) Diagrams showing the results of the Trypan blue assay with the percentage of viable cells for VK2 E6/E7 chronically exposed to low and high concentrations of PE N/MPLs or vehicle for 21 days. (b) Illustrative images of vaginal keratinocytes treated with vehicle or PE 25–250 μg/mL at day 3 to 21 of exposure. Pictures were taken at 40× magnification. Scale bars are 50 μm. (c) Graphs for RT-qPCR analysis displaying a relative expression of proliferation/differentiation-associated genes in VK2 E6/E7 chronically exposed to vehicle or PE 25–250 μg/mL for 21 days. (d) Histograms showing relative fold regulation for VEGF concentrations detected in VK2 E6/E7 cell culture supernatants at 3 to 21 days of exposure to PE 25–250 μg/mL or vehicle. (e) Relative gene expression of epigenetic regulation enzymes in vaginal keratinocytes exposed to PE 25–250 μg/mL or vehicle for 21 days. (f) Representative images of colony formation assays of VK2 E6/E7 chronically exposed to PE 25 μg/mL or vehicle for one month and two weeks. Staining was performed with Crystal violet. Relative absorbance for solubilised Crystal violet was reported on a diagram. Statistical analyses were conducted by unpaired, two-tailed Student’s t-test (n = 3) with “ns” non-significant, * p ≤ 0.05 and ** p ≤ 0.005.