Figure 4.

Nonautonomous induction of endothelial NF-κB signaling regulates senescence surveillance and specific immunocyte recruitment. (A) Experimental setup: Cdh5-Cre:ERT2; Rela^fl/fl or Rela^{wild-type (WT)} mice underwent hydrodynamic tail vein (HDTV) injection with NRAS^G12V-containing transposons and then intraperitoneal injection with tamoxifen (Tam), leading to deletion of Rela in endothelial cells. (B) LSECs were harvested 6 d after HDTV injection and analyzed for the indicated gene expression by qPCR. Dots are individual mice, and bars are means. Data were analyzed by unpaired Student's t-test; (*) P ≤ 0.05, (***) P ≤ 0.001. (C) Experimental setup: Cdh5-Cre:ERT2; Rela^fl/fl or wild-type mice underwent hydrodynamic tail vein (HDTV) injection with transposons containing oncogenic NRAS^G12V and then intraperitoneal injection with tamoxifen (Tam) before harvesting at 12 d after HDTV injection and analysis of RIS hepatocytes by immunohistochemistry. (D,E) Representative photomicrographs of RAS immunochemistry from the indicated conditions (D; scale bar, 200 µm) with quantification (E). Dots are individual mice, and bars are means. Data were analyzed by unpaired Student's t-test; (**) P ≤ 0.01. (F) Experimental setup: Cdh5-Cre:ERT2; Rela^fl/fl mice underwent hydrodynamic tail vein (HDTV) injection with NRAS^G12V-containing transposons and intraperitoneal injection with tamoxifen (Tam) or vehicle control before harvesting at 9 d after HDTV injection. Intrahepatic CD4⁺ T lymphocytes were flow-sorted before droplet-based scRNA sequencing. (G,H) tSNE clustering of 8152 CD4⁺ T cells from four mice in each indicated condition, showing differential abundance of cells in different clusters (G) and log₂ normalized Stat1 expression across all eight mice (H). (I) Expression plot showing cluster-specific expression of the indicated interferon-stimulated genes in the indicated CD4⁺ T-cell clusters from scRNA-seq data. (J) Representative immunofluorescence of liver sections from mice (as in F) injected with the indicated constructs, showing Stat1 expression in CD4⁺ cells (arrowheads). Scale bar, 20 µm. n = 3 biological replicates. (K) Flow cytometric analysis of Stat1 phosphorylation at Ser727 in intrahepatic CD4⁺ T lymphocytes in the indicated conditions. Dots are individual mice, and bars are means. Data were analyzed by one-way ANOVA with Sidak's multiple comparisons test.