Fig. 6: ssrA2-tagging of STING to photo-manipulate innate immune response.

a Schematic demonstrating light-dependent activation of STING signaling via coupling PhoBIT2 with cytosolic STING (cSTING; aa 152–379). Light-dependent oligomerization of sspB2-CRY2 leads to co-clustering of ssrA-cSTING, which in turn activates downstream TBK1 and IRF3 phosphorylation. Next, phosphorylated IRF3 (p-IRF3) undergoes nuclear translocation to trigger type I interferon responses. b Domain architectures of PhoBIT2-cSTING variants used in the study. Part A consists of GFP control, GFP-cSTING, either tagged or untagged with ssrA at the N and/or C termini. Part B features mCherry, CRY2, and sspB2 arranged in two different configurations. c Heatmap showing the degree of oligomerization by measuring the fold change in GFP intensity (Part A) relative to mCherry (Part B) following light stimulation. d Confocal images of HeLa cells co-expressing the indicated constructs before and after photostimulation. Scale bar, 10 μm. e The intensity profiles of GFP-ssrA2-cSTINGct-ssrA2 (A4; green) and sspB2-mCh-CRY2 (B2; red; across the red line) in response to photostimulation. f Western blot analysis of TBK1, IRF3, and their phosphorylated forms, in A549 cells expressing the indicated constructs before and after photostimulation. GAPDH is used as the loading control. g ELISA measurements of secreted IFN-β. A549 cells expressing the indicated constructs were either kept in the dark or exposed to pulsed blue light (20 s ON and 30 s OFF for 8 h). Data are shown as mean ± s.e.m from three biological replicates. Statistical analysis was performed using two-way ANOVA followed by Turkey’s post hoc test. Polyinosinic:polycytidylic acid (Poly I:C) is a synthetic double-stranded RNA (dsRNA) analog that acts as an immunostimulant in the assay. (Source Data).