Loss of Zhx2 promotes NK cell survival. (A–D) FCM analysis of EdU (A and B) and Ki67 (C and D) levels in Zhx2^+/+ and Zhx2^Δ/Δ splenic NK cells cultured for 12 h with 5 ng/ml IL-15 (representative of three independent experiments). A and C are representative FACS plots (left) and summary data (right) showing the percentage of EdU⁺ NK cells (A) and Ki67⁺NK cells (C). B and D show EdU⁺ level (B) and Ki67⁺ level (D) in NK subsets determined by CD11b/CD27 of the Zhx2^+/+ and Zhx2^Δ/Δ groups. DN, double negative; FSC, forward scatter. (E and F) FACS plots and graphs show NK cell viability determined by annexin V/7-AAD staining (representative of at least two independent experiments; n = 4 in F). Splenic NK cells from Zhx2^+/+ and Zhx2^Δ/Δ mice were either ex vivo assayed (E) or pretreated with CHX for the indicated times (F). (G) GSEA of Zhx2^+/+ and Zhx2^Δ/Δ NK cell bulk RNA-seq data. The normalized enrichment score (NES) and FDR are shown. (H and I) NK cells isolated from Zhx2^+/+ and Zhx2^Δ/Δ mice were labeled with CTV or CFSE and adoptively transferred into C57BL/6 hosts (H) or NSG hosts (I). FACS plots and graphs illustrate the percentages and the ratios of Zhx2^+/+ and Zhx2^Δ/Δ NK cells at the indicated time points, with day 0 as the time of adoptive transfer (representative of at least two independent experiments). (J) FACS plots show annexin V/7-AAD staining on CD27⁻CD11b⁺ NK subsets. Graphs depict percentages of early and late apoptosis (representative of three independent experiments). Dots represent data from individual mice, and error bars represent SEM per group in one experiment. Data were analyzed using Student’s t test (two-tailed paired t test). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.