Figure 4.

PI4KB and OSBP inhibition have opposite effects on STING activation

A. THP-1 cells were pre-incubated with the PI4KB inhibitor BF738735 and subsequently stimulated with the STING agonist 2’3’-RR CDA and tdTomato-reporter expression was quantified by flow cytometry. Mean ± SEM of n = 3 biological replicates out of 3 independent experiments are shown. A paired one-tailed t-test was performed on data that was not normalized to the controls.

B. PI4KB mRNA expression levels in THP-1 cells expressing a control gRNA or gRNAs targeting PI4KB. Mean ± SEM of n = 4 biological replicates out of 4 independent experiments are shown. One-sample t-tests were performed to compare each group to the normalized control value set at 1.

C. Cells in (b) were stimulated with 2’3’-RR CDA and tdTomato reporter expression was quantified by flow cytometry. Mean ± SEM of n = 5 biological replicates out of 5 independent experiments are shown. Statistical tests were performed on unnormalized data. We performed paired one-way ANOVA followed by Dunnett’s multiple comparisons post-tests to compare each treatment group to the control group.

D. 293T cells expressing eGFP-mSTING were transfected with a phosphatase-dead Sac1 (inactive), active Sac1 wt, or Sac1-kkaa mutant and stimulated or not with 2’3’-RR CDA (RR-CDA). After stimulation, cells were stained for phospho-STING and analyzed by flow cytometry. Mean ± SEM of n = 4 biological replicates out of 4 independent experiments are shown. One-sample t-tests were performed to compare each group to the normalized control value set at 100.

E. THP-1 cells were preincubated with DMSO or the OSBP inhibitors itraconazole (ITZ) or OSW-1 and stimulated with 2’3’-cGAMP or left untreated. Reporter expression was quantified by flow cytometry. Representative images of n = 3 biological replicates out of 3 independent experiments are shown.

F. Mean fluorescence intensity of tdTomato reporter in THP-1 cells stimulated with 2’3’-cGAMP in the presence of DMSO, itraconazole (ITZ), or OSW-1. Mean ± SEM of n = 3 biological replicates out of 3 independent experiments are shown. Statistical tests were performed on unnormalized data. We performed paired one-way ANOVA followed by Dunnett’s multiple comparisons post-tests to compare each treatment group to the control group.

G. CXCL10 mRNA levels in THP-1 cells pre-treated with DMSO or itraconazole (ITZ) and stimulated with 2’3’-RR CDA (RR-CDA). Mean ± SEM of n = 3 biological replicates out of 3 independent experiments are shown. A paired one-tailed t-test was used to compare the ITZ group to the DMSO control group.

H. tdTomato reporter expression of THP-1 cells pre-treated with the PI4KB inhibitor (PI4KBi) BF738735 followed by pre-treatment with itraconazole (ITZ) and stimulation with 2’3’-cGAMP. Mean ± SEM of n = 3 biological replicates out of 3 independent experiments are shown. We performed paired one-way ANOVA followed by Dunnett’s multiple comparisons post-tests to compare the indicated treatment groups to the control group.

* P< 0.05, ** P< 0.01, *** P< 0.001.