Fig 2: Investigation of the uptake features of Fe3O4 NPs in Caco-2 cells. (A,B) The level of mRNA expression of clathrin heavy chain (CHC) and Cav-1; (C) The level of protein expression of CHC and Cav-1 detected by Western blotting (WB); (D,E) Impacts of various uptake inhibitors on the uptake of Fe3O4 NPs, quantitative amounts of remaining NPs in the cells. ** p <0.01, and *** p <0.001.

Fig 3: TBK1-dependent phosphorylation controls the binding of STING to AP-1.a, Schematic diagram of the CTT of human STING (hSTING) and sequence logo of the CTT as indicated from 50 species. b, HeLa STING-knockout (KO) cells transfected with Flag-tagged STING?C?? (?1–341), wild-type (WT) STING, STINGE(E360A), STINGLI(L364A/I365A) or STINGELI(E360A/L364A/I365A) were treated with diABZI for 2 h. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. c, WCL and extracted CCV fractions from HeLa STING KO cells reconstituted with Flag-tagged wild-type STING or STINGLI(L364A/I365A) and treated or not with diABZI were analysed by western blot. CHC was used as a loading control. d, Glutathione sepharose pull-down assays of wild-type LBD-STING or LBD-STINGELI by glutathione S-transferase (GST)-tagged AP-1 core with or without ARF1. e, HeLaSTING cells transfected with NC siRNA or siRNAs against TBK1 or IRF3 were treated with or without diABZI. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. GAPDH was used as a loading control. f, HeLa wild-type cells, HeLa TBK1 KO cells and HeLa IRF3 KO cells stimulated with diABZI for 0, 1, 2 or 4 h were analysed by western blot. Ratios of target proteins versus loading control normalized to the 0-h time point of each condition. Vinculin was used as a loading control. g, Bio-layer interferometry binding studies of LBD-STING (top) or TBK1-phosphorylated LBD-STING (pSTING) (bottom) with AP-1 ?µCTD. The right graphs show the binding affinity of STING (top) and pSTING (bottom). One representative example of at least three (b,d,f) or two (c,e,g) independent experiments is shown.Source Data

Fig 4: AP-1 loading of STING into CCVs at the TGN.a, Whole-cell lysate (WCL) and CCV fractions from HeLa cells expressing Flag-tagged STING (HeLaSTING) were analysed by western blot. Clathrin heavy chain (CHC) and GAPDH were used as loading and processing controls. b, Airyscan imaging of HeLaSTING cells stimulated with diABZI. One representative cell of n = 7 cells. White arrows point at occurrences of pSTING. Scale bars, 4 µm (left) or 1 µm (magnified panels). c, Quantification of the colocalization of pSTING with CHC described in b, using Manders’ colocalization coefficients. Mean ± s.e.m. of n = 7 cells. d, STED images showing pSTING enclosed in CCVs from cells transfected with mCherry–clathrin and Flag–STING and stimulated with diABZI. Regions 1 and 2 are magnified from a large-field-of-view STED image (see Extended Data Fig. 2a). Scale bars, 200 nm. e, CLEM of HeLaGFP–STING cells stimulated with diABZI. The images depict box 3 of Extended Data Fig. 2b in Airyscan microscopy (left) or electron microscopy at different Z-heights (three right panels). White arrows indicate CCVs. Scale bars, 0.5 µm. f, WCL and CCV fractions from HeLa cells that were treated with non-targeting control (NC) small interfering RNA (siRNA) or AP-1 siRNA and stimulated with diABZI were analysed by western blot. CHC was used as a loading control. g, Quantification of the area of pSTING in bright-field fluorescent microscopy images of HeLaSTING cells that were treated with siRNAs and stimulated with diABZI. Mean ± s.e.m. of n = 3 independent experiments with 99 fields of view per condition. h, HeLa cells transfected with siRNAs and stimulated with diABZI were analysed by western blot. Ratios of STING versus loading control (GAPDH) normalized to the 0-h time point of each condition. One representative example of three (a–c,h) or two (f) independent experiments is shown.Source Data

Fig 5: Endocytic mechanism of rVAR2. (a) Western blot for rVAR2, CHC, and Cav-1 in PC3 and COLO205 cells following transfection with siSCR, siCHC, or siCav-1. Actin was used as loading control. (b) Confocal images of rVAR2 in Triton X-100 treated PC3 and COLO205 cells, transferrin (TFn) was used as control for Triton X-100 treatment. Scale bar represents 10 µM. (c) Colocalization of rVAR-488 and Dextran-594 in COLO205 and PC3 cells at indicated timepoints. Scale bar represents 5 µM. (d) Electron microscopy of COLO205 cells treated with rVAR2. Gold-nanoparticle labeled antibodies was used to detect the presence of rVAR2. Arrowheads indicate the presence of rVAR2 binding near membrane protrusions that corresponds to structures formed during macropinocytosis.