STAT2 hinders STING intracellular trafficking and reshapes its activation in response to DNA damage

Significance Therapeutic failure is a major problem for cancer patients. Chemotherapy often induces DNA damage or arrest of DNA synthesis, leading to the abnormal presence of DNA in the cytoplasm, which activates the cGAS–STING pathway. STAT2 binds to STING, modulating its function by inhibiting the expression of IRF3-dependent antitumor genes, but not of NF-κB–dependent protumor genes, thus helping cancer cells to resist DNA damage. This function of STAT2 is dependent on its phosphorylation on T404, a high level of which is seen in about 25% of human lung cancer specimens. Our data reveal a mechanism, quite distinct from its regulation of transcription, through which STAT2 reshapes STING activation and promotes malignancy.

A B C

Fig. S2. Interaction with STING is essential for inhibition by STAT2
A. GST-pull-down assay, using HEK293T cells transfected with GST-tagged truncations of STING. B. GST-pull-down assay, using HEK293T cells transfected with the indicated GST-tagged deletion of STING. C. GST-pull-down assay, using HEK293T cells transfected with GST-tagged specific deletions of STING. D. GST-pull-down assay, using HEK293T cells transfected with GST-tagged WT and E316A mutant of STING. E. STING E316 and surrounding sequences across species. F. STING-deficient HT1080 cells restored with WT-or E316A-STING were infected with HSV-1 (MOI=5) for 1 h, the media were refreshed for 4 h, followed by RT-PCR analysis.

316
G. STING-deficient HT1080 cells restored with WT-or E316A-STING were infected with HSV-1 (MOI=5) for 1h, then the media were refreshed for 16h. The media were analyzed for HSV by plaque assay.

Fig. S3.
A. Calu-1 cells transfected transiently STAT2-STING-V155M BiFC were monitored by confocal fluorescence microscopy. B. Calu-1 cells were transfected with STAT2-GFP (green) for 24 h, treated with cGAMP for 1 h. The cells were fixed and stained with calnexin antibody (red). Imaging data from confocal A D C B E fluorescence microscopy were analyzed by Fuji software to reveal co-localization, shown as white dots. C. PBMC cells were cultured in RPMI-1640 medium and transfected with 8 µg/ml cGAMP for 1 h. The cells were fixed and then stained with the indicated antibodies and DAPI. Imaging data from confocal fluorescence microscopy were analyzed by Fuji software to reveal colocalization as white dots. D. IP analysis of STAT2-STING-calnexin interactions, in HT1080 and 293T cells transfected with STING-GFP for 48 h and treated with 8 µg/ml cGAMP for 3 h. E. GST-pull-down assay using HEK293T cells transfected with GST-tagged truncations of STAT2. ND: N-terminal Domain, CCD: Coil-coiled Domain, DBD: DNA-binding Domain, TAD: Trans-activation domain;

E F
D. STAT2-deficient Calu-1 cells restored with WT, T404A, or T404E STAT2 were stimulated with 2'3'cGAMP (8 μg/ml) for 3 h, followed by Western analysis E. Primary STING -/-MEF cells were transfected with the indicated plasmids for 24 h and treated with cGAMP for 1 h. The cells were fixed, then stained with HA (purple), CD63 (red), and IRF3 (green) antibodies and DAPI. Imaging data from confocal fluorescence microscopy were analyzed with Fuji software to reveal co-localization as white dots. T403 is the residue corresponding to human T404 in mouse STAT2. F. Primary MEF cells were transfected with the indicated plasmids for 24 h and treated with cGAMP for 1 h, followed by nuclear fractionation and analyzed by the Western method. A. Whole cell lysates of H196, A549, Calu-1 cells were analyzed by the Western method. B. A549 cells expressing empty vector (plv) or STAT2 cDNA were treated with cisplatin for 72 h and cell survival was determined by using the MTT assay. C. Calu-1 cells with or without STAT2 knockdown were treated with cisplatin for 72 h and cell survival was determined with the MTT assay. D. H196 cells with or without STAT2 knockdown were treated with cisplatin for 72 h and cell survival was determined with the MTT assay.