Journal: Nature Communications

Article Title: Cholesterol-binding motifs in STING that control endoplasmic reticulum retention mediate anti-tumoral activity of cholesterol-lowering compounds

doi: 10.1038/s41467-024-47046-5

Figure Lengend Snippet: a Immunoblot analysis of THP1 cells treated with increasing MβCD doses (0–2 mM) before cGAMP (50 µg/ml) stimulation for 1 h. Quantified data presented as mean ± SD from three independent experiments. b Immunoblot analysis of STING dimer and oligomer formation in THP1 cells treated with increasing doses of MβCD before stimulation with cGAMP (50 µg/ml, 1 h). ( n = 3 biologically independent experiments with similar results). c THP1 cells electroporated with Cas9 protein and gene-specific sgRNAs as indicated. Five days post-electroporation, cells stimulated with vehicle or cGAMP (50 µg/ml) for 1 h. Immunoblot analysis performed for indicated protein levels. Quantified data shown as mean ± SD from three independent experiments. d Immunoblot analysis of STING, SeC24 or TBK1 co-immunoprecipitated with either endogenous expressed STING or STEEP in THP1 cells. Prior to the immunoprecipitation cells were pre-treated with mock or MβCD (2 mM) and then stimulated with cGAMP (50 µg/ml). IgG from the Sheep/Rabbit serum was used as negative controls. ( n = 2 biologically independent experiments with similar results). e , f THP1-derived macrophages were treated with cGAMP (5 µM) with varying concentrations of MβCD ( e ) or treated with MβCD (1 mM) and varying amounts of cGAMP (5 µM) ( f ) for a total of 6 h. Cell supernatants were analyzed for IFNα/β by HEK-blue bioassay and CXCL10 expression using ELISA. g Flow cytometry analysis of expression levels of maturation markers HLA-DR, CD86, and CD83 in human DC treated with cGAMP (5 µM) with or without filipin-III (1 µg/ml) or methyl-β-cyclodextrin (MβCD) (1 or 2 mM). The experiments for ( a – d) were independently repeated three times with similar results. The representative data from one experiment is shown in the figure. The experiment for ( e , f , g ) represent one experiment (repeated twice) done in experimental triplicates shown as mean ± SD. Statistical significance in experiment ( e , f , g ) was assessed using one-way ANOVA with Tukey’s multiple comparison correction. (ns not significant; * p < 0.05; ** p < 0.01, *** p < 0.001).

Article Snippet: Antibodies used for flow cytometry analysis of murine BMDC maturation were rat IgG2b κ anti-mouse-MHC Class II (I-A/I-E)-PE (Thermo Fisher Scientific), rat IgG2a κ anti-mouse-CD86-eFlour 450 (Thermo Fisher Scientific), rat IgG2b κ isotype control PE (Thermo Fisher Scientific) and rat IgG2a κ isotype control eFlour 450 (Thermo Fisher Scientific).

Techniques: Western Blot, Electroporation, Immunoprecipitation, Derivative Assay, Bioassay, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Comparison