![Changes in cell volume drive IFNβ responses through a DNA- and TBK1-dependent pathway. (A and B) Western blot (A) and densitometry (B) of viperin in WT BMDMs incubated in iso-osmotic media (± the TLR3 agonist poly I:C [1 μg ml−1]) or in hypo-osmotic media (50% H2O vol/vol in DMEM) in the presence of the TBK1/IKKε inhibitor MRT67307 (MRT, 10 µM) or vehicle control (DMSO, 0.1% vol/vol) (6 h) (n = 5). (C and D) Western blot (C) and densitometry (D) of viperin in WT BMDMs incubated in hypo-osmotic media in the presence of BafA1 (100 nM) or vehicle control (DMSO, 0.5% vol/vol) (6 h) (n = 5). (E) Schematic of the cGAS-STING pathway and inhibitors used. A151 inhibits dsDNA sensing, Ru.251 and 4-sulfonic calix[6]arene are inhibitors of cGAS, and H151 inhibits STING. (F and G) qRT-PCR analysis of Ifnb (F) and Cxcl10 (G) in WT and VRAC KO BMDMs incubated in hypo-osmotic media (50% H2O vol/vol in DMEM) for 6 h in the presence of H151 (10 µM), A151 (1 µM), or vehicle control (DMSO 0.5% vol/vol) (n = 4). (H and I) qRT-PCR analysis of Ifnb (H) and Cxcl10 (I) in WT and VRAC KO BMDMs incubated in hypo-osmotic media for 6 h in the presence of Ru.251 (10 µM) or vehicle control (DMSO, 0.5% vol/vol) (n = 4). (J and K) qRT-PCR analysis of Ifnb (J) and Cxcl10 (K) in WT and VRAC KO BMDMs incubated in hypo-osmotic media for 6 h in the presence of 4-sulfonic calix[6]arene (30 µM) or vehicle control (DMSO 0.5% vol/vol) (n = 4). (L) IFNβ release in the supernatant from WT and VRAC KO BMDMs stimulated with transfected poly dA:dT (1 μg ml−1), mock-transfected, or treated with CMA (250 μg ml−1) for 6 h (n = 5). (M) qRT-PCR analysis of Sting1 in WT and VRAC KO BMDMs treated with siRNA targeting Sting1, or nontargeting, before incubation in iso-osmotic or hypo-osmotic media (6 h) (n = 3). (N) qRT-PCR analysis of Cgas in WT and VRAC KO BMDMs treated with siRNA targeting Cgas, or nontargeting, before incubation in iso-osmotic or hypo-osmotic media (6 h) (n = 3). (O and P) qRT-PCR analysis of Ifnb (O) and Cxcl10 (P) in BMDMs from M and N (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, determined by a two-way ANOVA with Sidak’s post hoc analysis (2B, 2D, 2M, and 2N) or Dunnett’s post hoc analysis (2F–2K, 2O, and 2P). Values shown are the mean ± the SEM. Source data are available for this figure: SourceData F3. Refer to the image caption for details.](https://cdn.rupress.org/rup/content_public/journal/jcb/225/6/10.1083_jcb.202411133/1/jcb_202411133_fig3.png?Expires=1781198940&Signature=uppk4AD5boMGiAFCAP-wJiAjwWS6-zZ00QJRZIgS3rGVu9lfjy08aXoiz6HcD8Bmz3myp4cU43x-skAPZLOOuFilAqgE0rPrgtby7qp50HAE-46Ptkxuu1xhWCfpCSjLHtBO-mN5krWGY8WWHW6goRflPaALdV2Y8e81xP7bkvxSWjovJ1SeZnbdT5Yw~AUcA2-b6PrAYB8EXBZKU~peu1mXhfZoiGyBZZjyCtImQAfaDhCcf~4FYUH31sPoIHemz5f0TI-ULPMdFuEhUug48XsN3AqirZOaPXkJ0KQ7Cex4yN7HOXeHmnRCozncP3ePABr72Fibbk2HAFL5BVwbRQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Panel A: A western blot showing viperin and beta-actin levels in wild-type bone marrow-derived macrophages (WT BMDMs) under different conditions. The conditions include iso-osmotic media, the TLR3 agonist Poly I:C, and hypo-osmotic media, with and without the TBK1/IKK inhibitor MRT67307. Panel B: A scatter bar plot showing densitometry analysis of viperin levels from Panel A. The y-axis represents viperin densitometry, and the x-axis shows different treatment conditions. Panel C: A western blot showing viperin and beta-actin levels in WT BMDMs under hypo-osmotic media with and without bafilomycin A1. Panel D: A scatter bar plot showing densitometry analysis of viperin levels from Panel C. The y-axis represents viperin densitometry, and the x-axis shows different treatment conditions. Panel E: A schematic diagram illustrating the cGAS-STING pathway and the inhibitors used. Panel F: A scatter bar plot showing qRT-PCR analysis of Ifnb expression in WT and VRAC KO BMDMs under hypo-osmotic media with different inhibitors. The y-axis represents fold expression, and the x-axis shows different treatment conditions. Panel G: A scatter bar plot showing qRT-PCR analysis of Cxcl10 expression in WT and VRAC KO BMDMs under hypo-osmotic media with different inhibitors. The y-axis represents fold expression, and the x-axis shows different treatment conditions. Panel H: A scatter bar plot showing qRT-PCR analysis of Ifnb expression in WT and VRAC KO BMDMs under hypo-osmotic media with Ru.251. The y-axis represents fold expression, and the x-axis shows different treatment conditions. Panel I: A scatter bar plot showing qRT-PCR analysis of Cxcl10 expression in WT and VRAC KO BMDMs under hypo-osmotic media with Ru.251. The y-axis represents fold expression, and the x-axis shows different treatment conditions. Panel J: A scatter bar plot showing qRT-PCR analysis of Ifnb expression in WT and VRAC KO BMDMs under hypo-osmotic media with 4-sulfonic calix[6]arene. The y-axis represents fold expression, and the x-axis shows different treatment conditions. Panel K: A scatter bar plot showing qRT-PCR analysis of Cxcl10 expression in WT and VRAC KO BMDMs under hypo-osmotic media with 4-sulfonic calix[6]arene. The y-axis represents fold expression, and the x-axis shows different treatment conditions. Panel L: A scatter bar plot showing IFN-beta release in the supernatant from WT and VRAC KO BMDMs under different stimulation conditions. The y-axis represents IFN-beta release in ng/mL, and the x-axis shows different treatment conditions. Panel M: A scatter bar plot showing qRT-PCR analysis of Sting1 expression in WT and VRAC KO BMDMs treated with siRNA targeting Sting1 or non-targeting siRNA under different osmotic conditions. The y-axis represents fold expression, and the x-axis shows different treatment conditions. Panel N: A scatter bar plot showing qRT-PCR analysis of Cgas expression in WT and VRAC KO BMDMs treated with siRNA targeting Cgas or non-targeting siRNA under different osmotic conditions. The y-axis represents fold expression, and the x-axis shows different treatment conditions. Panel O: A scatter bar plot showing qRT-PCR analysis of Ifnb expression in BMDMs from Panels M and N. The y-axis represents fold expression, and the x-axis shows different treatment conditions. Panel P: A scatter bar plot showing qRT-PCR analysis of Cxcl10 expression in BMDMs from Panels M and N. The y-axis represents fold expression, and the x-axis shows different treatment conditions.
Changes in cell volume drive IFNβ responses through a DNA- and TBK1-dependent pathway. (A and B) Western blot (A) and densitometry (B) of viperin in WT BMDMs incubated in iso-osmotic media (± the TLR3 agonist poly I:C [1 μg ml−1]) or in hypo-osmotic media (50% H2O vol/vol in DMEM) in the presence of the TBK1/IKKε inhibitor MRT67307 (MRT, 10 µM) or vehicle control (DMSO, 0.1% vol/vol) (6 h) (n = 5). (C and D) Western blot (C) and densitometry (D) of viperin in WT BMDMs incubated in hypo-osmotic media in the presence of BafA1 (100 nM) or vehicle control (DMSO, 0.5% vol/vol) (6 h) (n = 5). (E) Schematic of the cGAS-STING pathway and inhibitors used. A151 inhibits dsDNA sensing, Ru.251 and 4-sulfonic calix[6]arene are inhibitors of cGAS, and H151 inhibits STING. (F and G) qRT-PCR analysis of Ifnb (F) and Cxcl10 (G) in WT and VRAC KO BMDMs incubated in hypo-osmotic media (50% H2O vol/vol in DMEM) for 6 h in the presence of H151 (10 µM), A151 (1 µM), or vehicle control (DMSO 0.5% vol/vol) (n = 4). (H and I) qRT-PCR analysis of Ifnb (H) and Cxcl10 (I) in WT and VRAC KO BMDMs incubated in hypo-osmotic media for 6 h in the presence of Ru.251 (10 µM) or vehicle control (DMSO, 0.5% vol/vol) (n = 4). (J and K) qRT-PCR analysis of Ifnb (J) and Cxcl10 (K) in WT and VRAC KO BMDMs incubated in hypo-osmotic media for 6 h in the presence of 4-sulfonic calix[6]arene (30 µM) or vehicle control (DMSO 0.5% vol/vol) (n = 4). (L) IFNβ release in the supernatant from WT and VRAC KO BMDMs stimulated with transfected poly dA:dT (1 μg ml−1), mock-transfected, or treated with CMA (250 μg ml−1) for 6 h (n = 5). (M) qRT-PCR analysis of Sting1 in WT and VRAC KO BMDMs treated with siRNA targeting Sting1, or nontargeting, before incubation in iso-osmotic or hypo-osmotic media (6 h) (n = 3). (N) qRT-PCR analysis of Cgas in WT and VRAC KO BMDMs treated with siRNA targeting Cgas, or nontargeting, before incubation in iso-osmotic or hypo-osmotic media (6 h) (n = 3). (O and P) qRT-PCR analysis of Ifnb (O) and Cxcl10 (P) in BMDMs from M and N (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, determined by a two-way ANOVA with Sidak’s post hoc analysis (2B, 2D, 2M, and 2N) or Dunnett’s post hoc analysis (2F–2K, 2O, and 2P). Values shown are the mean ± the SEM. Source data are available for this figure: SourceData F3.