FIGURE 3.

cGAS co-localizes with dsDNA in a time-dependent manner, while STING resides in the ER and dissociates upon stimulation of the upstream pathway. pDCs were isolated from peripheral blood of healthy donors and transfected with FITC-conjugated ISD or ssDNA. Co-localization of DNA and cGAS was determined by Amnis ImageStream. (A) Schemata of identification of pDCs among PBMC population. Single cells are selected out of PBMCs, first by plotting the population with area vs. aspect ratio. Next, focused singlets were determined by plotting the single cells with area vs. side scatter. Finally, pDCs were identified out of focused singlets as CD123⁺BDCA2⁺ cells. (B) Sample histograms showing co-localization between cGAS and DNA by bright detail similarity score in time-dependent manner. (C) Representative image of a single cell showing cGAS and dsDNA co-localization. (D) Quantification of co-localization between cGAS and DNA by bright detail similarity score. One way ANOVA was performed between all groups, and then post-hoc T tests were performed. Each dot represents an individual experiment (n = 3). (E, F) pDCs were purified and transfected with unconjugated ISD or ssDNA for 2 hours, fixed overnight, permeabilized, and stained for Calnexin and STING. (E) Histogram of Calnexin and STING co-localization with or without DNA transfection (Data represents one of two independent experiment). (F) Representative images of single cells showing Calnexin and STING co-localization with or without DNA transfection.