Association of IFI16, H2B, BRCA1, cGAS and STING during KSHV infection.

<p>(A) For a double PLA reaction, two independent reactions were performed. In the first PLA reactions, uninfected and KSHV (KS) infected (4 h) HMVEC-d cells were immunostained using mouse (ms) anti-IFI16 and goat (g) anti-H2B antibodies, and detected by DUOLink green detection agent. Cells were washed, blocked and subjected to the second PLA reactions using goat anti-H2B and rabbit (rb) anti-STING antibodies and visualized with red detection agents. Green and red dots indicate the localization of IFI16-H2B and H2B-STING, respectively. (B-E) Similarly, double PLA for different combinations were performed using BRCA1 (ms) + H2B (g) and H2B (g) + STING (rb); IFI16 (ms) + H2B (rb) and H2B (rb) + cGAS (g); BRCA1 (ms) + H2B (rb) and BRCA1 (ms) + cGAS (g); and H2B (ms) + STING (rb) and STING (rb) + cGAS (g). Red arrows indicate yellow dots representing the associations of three different proteins together such as IFI16-H2B-STING (A), BRCA1-H2B-STING (B), IFI16-H2B-cGAS (C), BRCA1-H2B-cGAS (D) and H2B-STING-cGAS (E) in the cytoplasm of KSHV infected cells. Nuclei were stained with DAPI. (F and G) Effect of BRCA1, cGAS and H2B on the interaction of IFI16-STING during KSHV <i>de novo</i> infection. (F) WCL from HMVEC-d cells electroporated with siC, siBRCA1, sicGAS and siH2B for 48 h then infected or uninfected with KSHV for 4 h were western blotted with anti-BRCA1, cGAS and H2B antibodies. (G) The WCL lysates from panel F were IP-ed with anti-IFI16 antibody and immunoblotted for STING and H2B. The bottom panels showed H2B knockdown efficiency and STING as an input control. (H and I) Role of ASC in the association of STING with IFI16 and H2B during KSHV <i>de novo</i> infection. WCL from HMVEC-d cells electroporated with siC or siASC for 48 h followed by with and without KSHV infections (4 and 24 h) were IP-ed with anti-STING antibody and immunoblotted for IFI16, H2B and STING. The bottom panels show the knockdown efficiency of ASC along with equal inputs of IFI16, H2B and STING by WB.