Panel A shows a schematic diagram of the proposed role for VRAC in cGAMP transport, indicating how VRAC containing LRRC8A, LRRC8C, and LRRC8E can act as a conduit for 23cGAMP. Panel B is a scatter bar plot showing the number of mRNA transcripts of LRRC8 subunit genes in WT BMDMs from RNA-sequencing, with the y-axis labeled as Log2(TPM+1) and the x-axis listing different LRRC8 genes. Panels C and D are scatter bar plots showing qRT-PCR analysis of Ifnb and Cxcl10 in WT and VRAC KO BMDMs, and L929 mouse fibroblasts, following incubation in isotonic or hypotonic media with or without extracellular cGAMP. The y-axis represents fold expression, and the x-axis lists different conditions. Panel E is a scatter bar plot showing qRT-PCR analysis for Ifnb in WT and VRAC KO BMDMs incubated in iso- or hypo-osmotic media in the presence of CMA or vehicle control. The y-axis represents fold expression, and the x-axis lists different conditions. Panel F is a western blot image showing phosphorylated and total IRF3 and TBK1 in WT and VRAC KO BMDMs under various conditions. Panels G and H are scatter bar plots showing densitometry of pIRF3 and pTBK1 from Panel F, with the y-axis representing the ratio of phosphorylated to total protein and the x-axis listing different conditions. Panel I is a semi-native PAGE image assessing STING dimerisation in WT BMDMs under different conditions. Panel J is a scatter bar plot showing densitometry of STING dimers from Panel I, with the y-axis representing dimer density and the x-axis listing different conditions. Panel K is a scatter bar plot showing IFN-beta release in the supernatant of WT and VRAC KO BMDMs under different conditions, with the y-axis representing IFN-beta concentration in ng/mL and the x-axis listing different conditions. Panels L and M are scatter bar plots showing qRT-PCR analysis for Cxcl10 and Rsad2 from the experiment in Panel E, with the y-axis representing fold expression and the x-axis listing different conditions. Panel N is a western blot image showing viperin in WT and VRAC KO BMDMs under different conditions. Panel O is a scatter bar plot showing IFN-beta release in WT and VRAC KO BMDMs under different conditions, with the y-axis representing IFN-beta concentration in ng/mL and the x-axis listing different conditions.
Cell volume disturbances and loss of VRAC potentiate STING signaling independent of cGAMP transport. (A) Schematic of the proposed role of VRAC in cGAMP transport. VRAC containing LRRC8A, LRRC8C, and LRRC8E can act as a conduit for 2′3′-cGAMP allowing transport between extracellular space. (B) Number of mRNA transcripts of LRRC8 subunit genes in WT BMDMs from RNA-seq in Fig. 1 A (TPM = transcript per million). (C and D) qRT-PCR analysis of Ifnb (C) and Cxcl10 (D) in WT and VRAC KO BMDMs, and L929 mouse fibroblasts, following incubation in isotonic or hypotonic media with or without extracellular cGAMP (5 µg ml-1) for 3 h (n = 4 for BMDMs, n = 3 for L929 s). (E) qRT-PCR analysis for Ifnb of WT and VRAC KO BMDMs incubated in iso- or hypo-osmotic media in the presence of the direct murine STING agonist CMA (250 μg ml−1) or vehicle control (1% DMSO vol/vol) (6 h) (n = 4). (F) Western blot (F) of phosphorylated (p-) and total IRF3 and TBK1 in WT and VRAC KO BMDMs incubated in iso-osmotic or hypo-osmotic media in the presence of CMA (250 μg ml−1) and or BFA (10 μg ml−1) or vehicle control (DMSO, 0.2% vol/vol) (3 h) (n = 6). (G and H) Densitometry of pIRF (G) and pTBK1 (H) from F (n = 6). (I) STING dimerization assessed by semi-native PAGE in WT BMDMs incubated in iso-osmotic or hypo-osmotic media in the presence of CMA (250 μg ml−1) or vehicle control (DMSO, 1% vol/vol) (4 h) (n = 6). (J) Densitometry of STING dimers in I (n = 6). (K) IFNβ release in the supernatant of WT and VRAC KO BMDMs incubated in iso-osmotic or hypo-osmotic media in the presence of CMA (250 μg ml−1) or vehicle control (DMSO, 1% vol/vol) and or BFA (10 μg ml−1) (6 h) (n = 6). (L and M) qRT-PCR analysis for Cxcl10 (L) and Rsad2 (M) from experiment in E. (N) Western blot of viperin in WT and VRAC KO BMDMs incubated in BMDMs in iso-osmotic or hypo-osmotic media in the presence of CMA (250 μg ml−1) and or BFA (10 μg ml−1) or vehicle control (DMSO, 0.2% vol/vol) (6 h) (n = 6). (O) WT and VRAC KO BMDMs were incubated in iso- or hypo-osmotic media + CMA (250 μg ml−1) for 3 h (first), before the media were replaced with fresh iso- or hypo-osmotic media (second) without CMA for a further 3 h. IFNβ release shown is from the second media incubation (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, determined by a one-way ANOVA with Dunnett’s post hoc analysis (6J) or two-way ANOVA with Dunnett’s post hoc analysis (6C and 6D), Sidak’s post hoc analysis (6E, 6 L, and 6M), or Tukey’s post hoc analysis (6F-6 G, 6K, and 6O). Values shown are the mean ± the SEM. BFA, brefeldin A. Source data are available for this figure: SourceData F6.