Journal: The EMBO Journal

Article Title: Termination of STING responses is mediated via ESCRT ‐dependent degradation

doi: 10.15252/embj.2022112712

Figure Lengend Snippet: A Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 4.5 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown are representative of three independent experiments. B Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated or treated with 25 μg/ml DMXAA for 1, 2 or 4 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown are representative of three independent experiments. C Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated or treated with 10 μg/ml 2′3′‐cGAM(PS)2 for 4 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown are representative of three independent experiments. D Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated (UT) or treated with 25 μg/ml DMXAA for 1, 2 or 4 h. Cells were fixed and stained for p‐STING (white) and the nucleus (DAPI; blue), before Z stack images were acquired on the LSM980 confocal microscope. Images are displayed as a maximum intensity projection (MIP) of Z stack images. Scale bar = 5 μm. Data shown are representative of three independent experiments. E, F The size (i.e. mean radius) (E) and mean intensity (F) of individual p‐STING punctate regions was quantified using CellProfiler. Data are shown as mean ± SEM combined from N = 3 independent experiments. Statistical analysis was performed using two‐way ANOVA using Bonferroni's multiple comparisons test, where * P < 0.05, **** P < 0.0001. A representative experiment used for quantification is shown in (D). G–I Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated (UT) or treated with 25 μg/ml DMXAA or 10 μg/ml 2′3′‐cGAM(PS)2 for 4 h. Cells were lysed for RNA purification and the expression of Ifnb1 (G), Isg15 (H) and Il6 (I) was analysed by qPCR. Data are shown as mean ± SEM combined from N = 3 independent experiments. Statistical analysis was performed using two‐way ANOVA using Bonferroni's multiple comparisons test, where * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. J Sting −/− iBMDMs expressing either WT hSTING or hSTING‐N154S were left untreated or treated with 1 μg/ml doxycycline (Dox) for 2 h. Cells were then washed and left to recover for a further 8 h in the absence or presence of 2 μM TAK243. Cells were lysed for immunoblot with indicated antibodies. Data shown are representative of three independent experiments. Source data are available online for this figure.

Article Snippet: DMSO (Sigma‐Aldrich #D2650), DMXAA (SelleckChem #S1537), 2′3′‐cGAM(PS)2 (InvivoGen #tlrl‐nacga2srs), LPS‐B5 Ultrapure (InvivoGen #tlrl‐pb5lps), Pam3CSK4 (InvivoGen #tltl‐pms), TAK243 (SelleckChem #S8341), H‐151 (InvivoGen #inh‐h151), doxycycline (Sigma‐Aldrich #D9891).

Techniques: Control, Western Blot, Staining, Microscopy, Purification, Expressing