Journal: EBioMedicine

Article Title: A STING antagonist modulating the interaction with STIM1 blocks ER-to-Golgi trafficking and inhibits lupus pathology

doi: 10.1016/j.ebiom.2021.103314

Figure Lengend Snippet: ISD017 associates with STING and STIM1 and prevents ER-to-Golgi Trafficking . (a) PMA-differentiated THP1 cells were treated with ISD017 (200 µg/ml). One h later, the cells were transfected with dsDNA (2 µg/ml). Cells were lysed at the indicated time points post-stimulation and immunoblotted by antibodies against pTBKs (S172), pSTING (S366), pIRF3 (S396), and Vinculin. ( n = 3). (b) PMA-differentiated THP1 cells were pretreated with ISD017 (200 µg/ml) for 1 h. The cells were transfected with dsDNA (6 µg/ml) and fixed 2 h later for imaging analysis after staining with anti-STING, anti-Calreticulin (ER), and anti-GM130 (Golgi) ( n = 3). Scale bar, 10 µm. (c) Lysates from PMA-differentiated THP1 cells were incubated with biotinylated ISD017, washed, and precipitated with streptavidin beads. Precipitates were immunoblotted with antibodies binding the indicated proteins ( n = 3). The light blue color of the bands for STEEP and vinculin in the input lane illustrates saturation of the signal. (d) Lysates from PMA-differentiated Wt, STING KO, and STIM1 KO THP1 cells were incubated with biotinylated ISD017, washed, and precipitated with streptavidin beads. Precipitates were immunoblotted with antibodies against STIM1, STING, and vinculin ( n = 3). (e) PMA-differentiated THP1 cells were treated with ISD017 (200 µg/ml) for 1 h, followed by stimulation with dsDNA for 2 hs. Cell lysates were subjected to immunoprecipitation with anti-STING and immunoblotting with anti-STIM1, anti-STING, and anti-vinculin ( n = 3). (f,g) Wt and STIM1 KO THP1 macrophages were treated with dsDNA in the presence or absence of ISD017 (200 µg/ml, pre-treatment). Supernatants were harvested 16 h post-stimulation, and IFN-I activity and TNFα levels were measured ( n = 3). The data in panel f and g are presented as means of biological triplicates +/- st.dev. [Statistical analysis of the data in f, g were performed using two-tailed one-way ANOVA test].

Article Snippet: The following antibodies and dilutions were used for immunoblotting anti‐STING (Cell Signaling, D2P2F/#13,647, 1:1000), sheep anti‐STING (R&D Systems, AF6516, 1:500), rabbit anti‐phosphoSTING (S366) (Cell Signaling Technology, #85,735, 1:1000), rabbit anti‐TBK1 (Cell Signaling, 3504, 1:1000), rabbit anti‐phospho-TBK1 (Ser172) (Cell Signaling, D52C2/#5483, 1:1000), IRF3 (Cell signaling, 11,904, 1:1000), phospho-IRF3 (S396) (Cell Signalling, 4947,1:1000, mouse anti‐p62 (Cell Signaling, # 88,588, 1:1000), anti-STEEP (Proteintech, 24,021–1-AP, 1:1000), anti-STIM1 (D88E10, Cell Signaling, #5668, 1:1000), rabbit anti‐LC3 (Cell Signaling, #3868, 1:1000), rabbit anti-cleaved caspase3 (Cell Signaling, #9664,1:1000), and mouse anti‐vinculin (Sigma, #V9131, 1:10,000).

Techniques: Transfection, Imaging, Staining, Incubation, Binding Assay, Immunoprecipitation, Western Blot, Activity Assay, Two Tailed Test