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. 2023 Feb 4;14:611. doi: 10.1038/s41467-023-36132-9

Fig. 3. An ESCRT complex containing HGS and VPS37A regulates STING degradation and signaling shutdown.

Fig. 3

a Enrichment heat-map of selected proteins. b mNG levels in 293 T STING-mNG cell lines KO for the indicated genes stimulated with 1 µg/ml 2′3′-cGAMP(pS)2 (in medium) for 24 h. One representative plot of n = 2 independent experiments with n = 2 technical replicates per experiment. c Percentage of STING-mNG positive cells in cells stimulated as in b. Shown is ratio %STING-mNG positive of each sgRNA over %STING-mNG positive cells of the control non-targeting sgRNA (ntgRNA). n = 2 independent experiments with n = 2 technical replicates per experiment. Each dot represents an individual replicate. One-way ANOVA with Dunnet’s multiple comparison test. d Immunoblot of the indicated proteins in 293T STING-TurboID stimulated with 2 µg/ml cGAMP (in perm buffer) for the indicated times, in the input and after streptavidin pulldown (PD: Strept.). One representative blot of n = 3 independent experiments. e Immunoblot of the indicated proteins in the input and post HA co-immunoprecipitation in 293T cells stably transduced with the indicated constructs. One representative blot of n = 3 independent experiments. f Same as in e for VPS37A. g Immunoblot of the indicated proteins in the input and post HA co-immunoprecipitation in 293T cells stably transduced STING-HA and stimulated with 2 µg/ml cGAMP (in perm buffer) for 3 h. One representative experiment of n = 2 independent experiments. h Immunoblot of the indicated proteins in BJ1 fibroblasts KO for HGS. Cells were stimulated with 0.5 µg/ml cGAMP (in perm buffer) for the indicated times. One representative blot of n = 3 independent experiments. i Same as in h) for VPS37A. j qPCR for IFNβ (left) and IL6 (right) in BJ1 fibroblasts KO for HGS (blue) or VPS37A (red) stimulated with 0.5 µg/ml cGAMP (in perm buffer) for 8 h. n = 3 independent experiments. 2−ΔCt Fold Change calculated as ratio 2−ΔCt sgRNA/2−ΔCt ntgRNA for cells stimulated with cGAMP. One-way ANOVA on log-transformed data with Dunnet multiple comparison test. In all panels, bar plots show mean and error bars standard deviation. Marker unit for Westen blots is KDa. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant.