Figure 3.

Analysis of the effects of PE N/MPL acute exposure on VK2 E6/E7 cytoskeleton and cell stress pathways. (a) Representative IF acquisitions showing actin cytoskeleton of vaginal keratinocytes treated with vehicle or PE N/MPLs at 25–250 μg/mL for 72 h. Actin is stained in red with DyLight™ 554 Phalloidin, and nuclei are stained in blue with DAPI. Arrowheads indicate gaps in the cytoskeletal net. Pictures were taken at 40× magnification. Scale bars are 50 μm. (b) Illustrative IF pictures showing β-Catenin localisation in VK2 E6/E7 exposed to vehicle or PE N/MPLs 25–250 μg/mL for 72 h. β-Catenin is stained in green with Alexa Fluor™ 488, while nuclei are stained in blue with DAPI. Acquisitions were obtained at 40× magnification. Scale bars are 50 μm. (c) Representative Western blot and corresponding densitometry for β-Catenin, E-cadherin, and Pan Keratin expression in vaginal keratinocytes exposed to vehicle or PE N/MPLs at 25–250 μg/mL for 72 h. β-Actin was used as a loading control (Figure S2). (d) RT-qPCR graphs showing relative expression of stress and redox homeostasis genes in VK2 E6/E7 exposed for 48 h to vehicle or PE 25–250 μg/mL. GAPDH mRNA level was used as endogenous control. (e) Histograms displaying epithelial barrier function-related microRNA expression in vaginal keratinocytes treated with vehicle or low and high PE particle concentrations for 48 h. U6 snRNA levels were employed as endogenous control. All the 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.