Figure 6.

Caraphenol A alters expression and subcellular localization of IFITM2/3 protein and late endosomes. (A) Representative images of HeLa cells obtained by confocal microscopy using 63× oil immersion objective lens at room temperature. HeLa cells were plated and treated for 4 hours with caraphenol A (30 μM), resveratrol (30 μM), or DMSO (0.06%) before addition of LV, MOI of 15, for 30 minutes or 2 hours. Cells were then fixed, permeabilized, and immunostained using α-IFITM2/3, α-LAMP1 antibodies, and Hoechst 33342 nuclei stain. Scale bars are 10 microns. Images were collected on Zeiss Zen Software and analyzed using Imaris InCell software, identifying the number and mean intensity of IFITM2/3 (B) and LAMP1 (C) stained vesicles per cell. At least 50 cells were imaged per condition and plotted as dot plots (caraphenol A, green symbols; resveratrol, orange symbols; DMSO, blue symbols; mean ± SD). *P < .032, **P < .0021, ***P < .0002, ****P < .0001 by Kruskal-Wallis test with Dunn’s multiple comparison correction. (D) Representative images of mPB-CD34⁺ HSPC that were thawed, prestimulated for 48 hours, and then either immediately imaged without LV and DMSO (0.06%) (top), or then treated for 4 hours with either DMSO (middle, 0.06%) or caraphenol A (bottom, 30 μM) before addition of LV for 30 minutes or 2 hours. Cells were fixed, stained, and analyzed as above for HeLa cells for IFITM2/3 (E), p24 protein (F), and LAMP1⁺ (G) vesicle number and mean vesicle intensity (mean ± SD).