Fig. 2: cGAMP triggers SOAT1-Mediated ER Cholesterol Reduction. | Nature Communications

Fig. 2: cGAMP triggers SOAT1-Mediated ER Cholesterol Reduction.

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

Fig. 2

a Colocalization analysis of ER and cholesterol in THP1 cells. Cells were probed with ER-Tracker™ Red and BODIPY-Cholesterol, then either unstimulated or stimulated with cGAMP (50 µg/ml) for 0, 2, or 4 h. Colocalization analyzed using ImageStream. Three cell examples without stimulation demonstrate cholesterol-ER colocalization. b Confocal microscopy of THP1 cells pre-stained with Bodipy 480 nm-Cholesterol and ER tracker, then untreated or treated with vehicle or cGAMP (50 µg/ml) for 1, 2, and 4 h. Images acquired using Zeiss LSM 800 confocal microscope, processed with Zen Blue software, and analyzed with ImageJ. c 15 images per group in (b) quantified using Pearson correlation coefficient tool in ImageJ. Box plot depicts data distribution. d THP1 cells electroporated with Cas9 protein and sgRNAs, then stimulated with 50 ug/ml cGAMP for 1 h. Phosphorylation levels of TBK, IRF3, and STING analyzed by immunoblotting. The sgRNA targeting the AAVS1 Safe-Harbor Site was used as control. The experiment was independently repeated three times with similar results. e Blot quantification data of (d) presented as mean ± SD from three independent experiments. f Colocalization analysis of ER and cholesterol in WT or SOAT1 KO THP1 cells using ImageStream. The cells were probed with ER-Tracker™ Red and BODIPY-Cholesterol, then stimulated with vehicle or cGAMP for 2 h. g Confocal microscopy of THP1 WT/ STING KO/ SOAT1 KO cells pre-stained with Bodipy 480nm-Cholesterol and ER tracker, then treated with vehicle or cGAMP for 2 h. Pearson correlation coefficient (r) quantification performed on 20 vehicle and 15 cGAMP-treated images per group. The experiments for (a, f) represent one experiment out of three independent experiments, with 10,000 cells per sample for colocalization percentage calculation. Quantitative results are presented as mean ± SD. Statistical analysis conducted with one-way ANOVA. Statistical analysis of (c, g) performed using one-way ANOVA with Tukey’s multiple comparison test. Representative data from one experiment shown (n = 2 biologically independent experiments with similar results). (ns not significant; *p < 0.05; **p < 0.01).

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