Zhx2 cell and subcell profiling. (A) Specific expression of ZHX1/2/3 in different immune cells. RNA-seq data are adopted from Gene Expression Omnibus accession no. GSE125188.(B) Expression of Zhx1/2/3 in NK cells isolated from indicated tissues. RNA-seq data are adopted from Gene Expression Omnibus accession no. GSE133383.BL, blood; LU, lung; SP, spleen. (C) Western blot detecting Zhx2 expression in purified splenic NK cells from Zhx2^+/+ and Zhx2^Δ/Δ mice. Actin was used as the loading control. (D and E) FACS plots and bar graphs represent percentages and absolute numbers of CD3⁻NK1.1⁺ NK cells in the liver, spleen, and blood from Zhx2^+/+ and Zhx2^Δ/Δ mice (representative of at least three independent experiments). (F) FACS plots and bar graphs depict NK cell subsets determined by expression of CD11b/CD27 in BM from Zhx2^+/+ and Zhx2^Δ/Δ mice (representative of three independent experiments). (G) NK cell counts in the indicated subsets from BM of Zhx2^+/+ and Zhx2^Δ/Δ mice (representative of three independent experiments). DN, double negative. (H) Western blot detecting Zhx2 expression in purified splenic NK cells from Cag-Zhx2^+/+ and Cag-Zhx2^Δ/Δ mice. Actin was used as the loading control. (I) FACS plots and bar graphs depict NK cell subsets determined by expression of CD11b/CD27 in the spleen from Cag-Zhx2^+/+ and Cag-Zhx2^Δ/Δ mice at day 0 before in vitro culture (representative of three independent experiments). (J) FCM analysis of NKP cells marked as Lin⁻CD27⁺CD244⁺CD122⁺NK1.1⁻. (K) NKP cell percentage and number in the spleen are shown (representative of two independent experiments). Each dot represents data from an individual mouse, and error bars represent SEM per group in one experiment. Data were analyzed using Student’s t test (two-tailed paired t test) in E–G, I, and K. *, P < 0.05; **, P < 0.0001; ***, P < 0.001; ****, P < 0.0001.