GoKawiilCell culture
HeLa (CCL-2) cells were obtained from Sigma-Aldrich. HEK293T cells were a gift from D. Trono, originally purchased from the American Type Culture Collection. BJ-5ta cells were obtained from the American Type Culture Collection. THP-1-Dual STING1 knockout cells were purchased from Invivogen (thpd-kostg). HeLa and HEK293T cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) (Thermo Fisher Scientific, 41965039) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco SKU, 10270106), 1% (v/v) penicillin (100 IU ml−1)–streptomycin (100 μg ml−1) (BioConcept, 4-01F00-H), 2 mM l-glutamine (Thermo Fisher Scientific, 25030024). BJ-5ta cells were cultured in a mixed medium consisting of 80% of DMEM and 20% of Medium 199 (Thermo Fisher Scientific, 41150087) supplemented with 10% FBS, 2 mM l-glutamine and 1% (v/v) penicillin–streptomycin. THP-1 cells were cultured in Roswell Park Memorial Institute 1640 Medium (RPMI 1640) (Thermo Fisher Scientific, 21875034) containing 10% (v/v) heat-inactivated FBS, 25 mM HEPES (BioConcept, 5-31F00-H), 2 mM l-glutamine and 1% (v/v) penicillin–streptomycin. All cells were cultured at 37 °C and at atmospheric O 2 and 5% CO 2 . HeLa cGAS knockout cells were reported previously38,61. Cell lines were routinely tested and confirmed to be negative for mycoplasma contamination.
Plasmids
The pEFBos-eGFP-STING and pEFBos-Flag-STING vectors were obtained by inserting STING, eGFP and Flag sequences flanked by 5′ XhoI and 3′ NotI sites into the pEFBos vector. The pEFBos-based STING mutations (I10E, P11E, R14E, R14W, G15I, K20I, E38K, E38R, Y46R, L54D, E68Q, E69L, E69N, R76E, S80I, L93Y, L100E, Y106K, Y106R, W119C, W119K/M120K, A142D, C148E, E149F, N154S, A156G, H157K, I165H, R169A, R169K, R169W, D205H, V208P, P209K, D210P, M214K, L268I, G278F, F279V, R284S, Q287S, D301W, P317R, D319G, D319K/D320K, D320K, E339K, E340K, E339K/E340K, S366A and P371G) were obtained by site-directed mutagenesis. The pSIN-based STING and STING mutations (W119K/M120K, I165H and D319K/D320K) were generated by Gateway cloning by means of the pDONR 221 vector into the pSIN vector. The primers used for plasmid are provided in Supplementary Table 1.
Generation of cell lines
To generate the IFN reporter cell line, the lentiviral plasmid41 (a gift from D. H. Raulet) encoding the tdTomato reporter gene driven by the IFN-stimulated response elements and the minimal mouse Ifnb promoter was transduced to HEK293T cells, followed by the selection under 100 μg ml−1 zeocin. For the eGFP-LC3B reporter, HEK293T cells were transduced with a pLVX lentiviral plasmid encoding eGFP-LC3B inserted between the 5′ XbaI and 3′ PmeI restriction sites, followed by fluorescence-activated cell sorting on the basis of eGFP signal.
To establish HEK293T cells, BJ-5ta cells and THP-1 cells with inducible expression of distinct STING mutants, corresponding cells were transduced with a pSIN lentiviral vector carrying eGFP-STING or STING alone and a puromycin-resistance gene. Cells were selected with puromycin (1 μg ml−1 for HEK239T and BJ-5ta, 2.5 μg ml−1 for THP-1).
Library design and construction
The library design was adapted from ref. 60. In brief, the Homo sapiens STING sequence was (codon-optimized and) mutated systematically on each amino-acid position into all the other 19 types of amino acid. The full-length sequence was split into three segments: residues 1–155, 156–270 and 271–379. Within each segment, only the indicated residues were mutated whereas the remainder of the coding sequence remained WT. These three sublibrary oligonucleotide pools were purchased from Twist Biosciences and amplified individually using Phanta polymerase (Vazyme, P515-00). PCR products were then inserted into the pSIN vector through Gateway cloning.
Library characterization and screening
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