Panel A shows a Western blot with bands representing protein samples from wild-type (WT) and VRAC knockout (KO) bone marrow-derived macrophages (BMDMs) treated with puromycin under different conditions. The blot is probed with anti-puromycin and anti-beta-actin antibodies. Panel B displays immunofluorescence images of WT and VRAC KO BMDMs stained for the stress granule marker G3BP1. Cells are shown under iso-osmotic and hypo-osmotic conditions, with stress granules indicated by arrowheads. Panels C, D, and E present scatter bar plots of qRT-PCR analysis. Panel C shows the fold expression of Mavs in WT and VRAC KO BMDMs treated with siRNA targeting Mavs or non-targeting siRNA under iso-osmotic and hypo-osmotic conditions. Panels D and E show the fold expression of Ifnb and Cxcl10, respectively, in the same BMDMs. The y-axes represent fold expression, and the x-axes categorize the treatments and conditions.
Changes in cell volume induce translation arrest and stress granule formation, but type I IFN signaling is independent of RNA sensing through MAVS. (A) Puromycin incorporation assay in WT and VRAC KO BMDMs incubated in DMEM, iso-osmotic media (50% PBS vol/vol in DMEM), hypo-osmotic media (50% H2O vol/vol/in DMEM), or CHXx (10 μg ml−1) (4 h) (n = 3). (B) Immunofluorescence for the stress granule marker G3BP1 in WT and VRAC KO BMDMs incubated in iso-osmotic or hypo-osmotic media (2 h) (n = 4) (scale = 20 µm; arrowheads indicate stress granule–positive cells). (C) qRT-PCR analysis of Mavs in WT and VRAC KO BMDMs treated with siRNA targeting Mavs, or nontargeting, before incubation in iso-osmotic or hypo-osmotic media (6 h) (n = 3). (D and E) qRT-PCR analysis of Ifnb (D) and Cxcl10 (E) in BMDMs from C (=3). ***P < 0.001, determined by a two-way ANOVA with Sidak’s post hoc analysis (4C) or Tukey’s post hoc analysis (4D and 4E). Values shown are the mean ± the SEM. CHX, cycloheximide. Source data are available for this figure: SourceData F4.