Figure 7.

The acute inflammatory environment in OTC+PBMC+IL cultures induces DNA damage and DNA strand breaks in the overlying epithelial cells. A: Representative low-power confocal microscopy images of nuclear DAPI (blue) and the DNA damage/DNA strands break marker γH2AX staining (red) in the squamous epithelium of OTC controls. Arrow indicates basal cell layer; arrowhead, superficial cell layers. Inset: High-power confocal image showing granular pattern of γH2AX nuclear staining in a rare positive cell. B: Same as in A except images of nuclear DAPI (blue) and γH2AX staining (red) in squamous epithelium from OTC+PBMC+IL (IL-2, -7, and -15) cultures. Arrow indicates basal cell layer; arrowhead, superficial cell layers. Inset: High-power confocal image showing granular pattern of γH2AX nuclear staining in multiple positive cells. C: Quantitative analysis of γH2AX⁺ cells. γH2AX⁺ cells were expressed as a percentage of the total number of DAPI-stained cells. D and E: Representative image from one of the experiments of stained nuclei from Comet assay for DNA strand breaks in overlying squamous epithelial cells from OTC control (D) or OTC+PBMC+IL (E) cultures. Nuclei with longer, brighter tails are an indicator of DNA strand breaks in Comet assays. F: Quantitation of Comet assay tail moment across the experiments. Data are expressed as means ± SD (C) and means ± SD of the mean (F). n = 3 cultures per condition, with at least 500 cells counted per culture (C); n = 3 cultures per condition, with 57 control and 50 PBMC+ILs Comet tails analyzed in total across the three cultures using OpenComet image analysis software version 1.3.0¹⁶ in total from the three experiments (F). ^∗P ≤ 0.05, U-test; ^∗∗∗∗P < 0.0001, unpaired t-test. Original magnification: ×200 (A and B, main images); ×900 (A and B, insets); ×100 (D and E). OTC, organotypic tissue culture; PBMC, peripheral blood mononuclear cell.