Dasatinib increases spleen stromal cell pMLC levels in a ROCK-dependent manner. (A) Flow cytometry gating strategy for the main stromal cells analyzed after 2-hour treatment with dasatinib (50 mg/kg) or DMSO. (B) Representative example of pMLC expression in BECs and FRCs. (C) Ratio of pMLC expression of stromal cell subpopulations from LNs and spleens of dasatinib- and control-treated mice. Analyzed by 1-sample t test. (D) Mean fluorescence intensity (MFI) of pMLC from BECs, marginal reticular cells (MRCs), and TRCs recovered from LNs and spleens of DMSO-treated mice. n = 5 mice per group from 4 independent experiments. (E) Representative confocal images of subconfluent FRCs cultured on 20 μg/mL of fibronectin and treated for 2 hours with DMSO, 100 nM dasatinib, 20 μM Y27632, or both inhibitors. Scale bars represent 100 μm. (F) Intensity of pMLC staining quantified by FIJI and normalized to the cell area. Each dot represents a cell. n = 3; analyzed by Kruskal-Wallis test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (G) Experimental layout for egress assay in combination with Y27632. (H) Percentage of CFSE⁺ B cells and CD4⁺/CD8⁺ T cells recovered from the spleens, normalized to DMSO-treated mice (dotted line) (left). FC of CFSE⁺ B cells and CD4⁺/CD8⁺ T cells in blood of mice treated with dasatinib (50 mg/kg) alone, or dasatinib and Y27632 (10 mg/kg), normalized to DMSO-treated mice (dotted line) (right). n = 9 mice per group, from 3 independent experiments; analyzed by 1-sample t test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Boxplots depict 25th and 75th percentiles and the median, and the whiskers show fifth and 95th percentiles. DN, CD31 PDPN double negative stromal cells; SPL, spleen.