Fig. 3: Depletion of cellular cholesterol facilitates STING trafficking from ER to Golgi. | Nature Communications

Fig. 3: Depletion of cellular cholesterol facilitates STING trafficking from ER to Golgi.

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

Fig. 3

a HEK293T cells with stable STING expression were treated with or without MβCD (2 mM) and then stimulated with vehicle or cGAMP (50 µg/ml) for 1 h. Fixed with 4% PFA, cells were stained with anti-PDI, anti-STING, and anti-GM130 to analyze STING trafficking from ER to Golgi via image stream. Three cell examples demonstrate the definition of cholesterol-ER colocalization. Quantitative results are presented as mean ± SD. Experiment included three sample replicates under the same conditions, with 10,000 cells per sample collected to calculate colocalization percentage. Statistical analysis performed as two-tailed unpaired t-test. b In vitro membrane budding assay containing cytosolic fractions from untreated THP1 cells and membrane fraction from microsomes extracted from THP1 cells treated with increasing doses of MβCD (0, 1, 2 mM) for 4 h. Budded materials were analyzed by immunoblotting to assess cholesterol depletion’s effect on membrane budding. “Membrane only” (M/O) served as a negative control. Experiment was independently repeated thrice with similar results. Blot quantification data presented as mean ± SD from three independent experiments. Statistical analysis conducted using one-way ANOVA with Tukey’s multiple comparisons test. c THP1 cells treated with or without MβCD (2 mM) and then stimulated with vehicle or cGAMP (50 µg/ml) for 1 h. Cells probed with anti-PDI (ER marker), anti-GM130 (Golgi marker), and anti-STING, analyzed using Zeiss LSM 800 confocal microscope. Images processed with Zen Blue software 3.8 (Zeiss) and analyzed with ImageJ. Ten images per group quantified through Pearson correlation coefficient (r) for STING-ER colocalization and Manders’ overlap coefficient for STING-Golgi colocalization in ImageJ. Box plot depicts data distribution. d THP1 cells treated with vehicle, MβCD (2 mM, 2 h), Cholesterol (5 mM, overnight), fixed with 4% PFA, stained with anti-Sec24 and anti-PDI. Samples subjected to Zeiss LSM 800 confocal microscope analysis. Ten images per group quantified for Sec24 foci using ImageJ. Statistical analysis of (c, d) conducted using Statistical analysis of (c, g) performed using one-way ANOVA with Tukey’s multiple comparison test; showing one representative dataset out of two biologically independent experiments, yielding similar results (ns not significant; *p < 0.05; **p < 0.01).

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