Fig. 1: Optogenetic modulation of the STING pathway using LiSmore.

a Schematic illustrating the comparison between physiological activation of the STING pathway upon cGAMP stimulation (left) and light-induced STING pathway activation using LiSmore (right). Photostimulation leads to CRY2-driven multimerization of the STING C-terminal tail (CTT), resulting in downstream TBK1 and IRF3 recruitment and phosphorylation. Phosphorylated IRF3 forms a dimer, translocates into the nucleus, and activates type I interferon responses. b Domain architectures of CRY2-pLxIS and its variants. The photolyase-homology region of CRY2 is fused with one or two copies of STING-CTT. The TBK1 and IRF3 docking sequences are shown in blue and green, respectively. c Confocal images of HeLa cells co-expressing mCh-CRY2-pLxIS (red) and TBK1-YFP (green) before and after photostimulation. The intensity profiles of TBK1-YFP (green) and CRY2-pLxIS (red; across the white line) in response to photostimulation are plotted on the right. Scale bar, 10 μm (1 μm in the magnification). Data are representative of three independent experiments. See Supplementary Movie 1. d Immunoblot analysis of TBK1, IRF3, and p65, as well as their phosphorylated forms, in THP-1 cells expressing mCh-CRY2-pLxIS before and after photostimulation at the indicated time points. Data are representative of three independent experiments. e, f Quantification of mRNA expression levels of the STING pathway-related signature genes by qRT-PCR, including RSAD2 (radical S-adenosyl methionine domain containing 2), CXCL10 (C-X-C motif chemokine ligand 10), IFNB (interferon beta 1), IFIT1-3 (interferon-induced protein with tetratricopeptide repeats 1, 2, and 3) and ISG15 (interferon-stimulated 15 kDa protein) (e) and secreted IFNβ with ELISA (f) in sorted mCherry⁺ HEK293T cells transfected with CRY2-pLxIS and CRY2-control. Cells were exposed to pulsed blue light (470 nm, 4 mW/cm², 30 s ON/OFF cycle for 8 h). n = 3 biological replicates; mean ± SD; Two-sided unpaired Student’s t-test. g Determination of secreted IFNβ using ELISA at varying light intensities. FACS-sorted HEK293T cells expressing the indicated constructs (mCherry⁺ ) were either kept in the dark or exposed to pulsed blue light delivered at 0.1, 0.5, 1, and 2 mW/cm² (30 s ON and 30 s OFF for 8 h). n = 4 biological replicates (mean ± SD); Two-sided unpaired Student’s t-test.