Panel A shows a line graph depicting the regulatory volume decrease measured by calcein fluorescence. The x-axis represents time in minutes, and the y-axis represents the fluorescence ratio (F slash F0). The graph shows that WT macrophages undergo a regulatory volume decrease, while VRAC KO macrophages do not. Panel B is a bar graph showing the area under the curve analysis of Panel A. The x-axis represents the genotype (WT and VRAC KO), and the y-axis represents the area under the curve. The bar graph indicates a significant difference between WT and VRAC KO macrophages. Panels C, D, and E are scatter plots comparing gene-wise Log2 fold change (Log2FC) values following hypotonic treatment in WT and KO BMDMs. Panel C is labeled by significance compared to respective isotonic controls, Panel D by significance in KO cells compared to WT under hypotonic conditions, and Panel E by the effect of VRAC KO on modifying the WT hypotonic effect. Panels F, G, and H are heatmaps displaying the expression of core enrichment genes from gene sets differentially regulated in KO vs WT cells following hypotonic treatment. The heatmaps are scaled by gene-wise Z-score, with the x-axis representing different genotypes and treatments, and the y-axis representing different genes. The heatmaps show distinct patterns of gene expression between WT and VRAC KO macrophages under different conditions.
R elated to Fig. 1: KO of LRRC8A removes RVD in BMDMs and further RNA-seq analysis. (A) RVD measured by calcein fluorescence in WT or VRAC KO BMDMs incubated in hypo-osmotic media (50% vol/vol H2O in DMEM, ∼170 mOsm kg−1), added at the indicated time point (n = 3). These data were obtained from the same cultures of BMDMs used for RNA-seq analysis in Fig. 1. (B) Area under the curve analysis of A. (C–E) Scatter plot comparing gene-wise Log2FC values following hypotonic treatment in WT and KO BMDMs, labeled by significance compared with respective isotonic controls (C), by significance in KO cells compared with WT under hypotonic conditions (D), and by the effect of VRAC KO on modifying the WT hypotonic effect (E). (F–H) Heatmaps displaying expression of the core enrichment genes (determined via GSEA) from gene sets differentially regulated in KO vs WT cells following hypotonic treatment, scaled by gene-wise Z-score. **P < 0.01, determined by an unpaired t test. Values shown are the mean ± the SEM. Log2FC, Log2 fold change.