TRIM13 nuclear localization is necessary for its antileukemic function. (A) Confirmation of TRIM13 antibody specificity (histogram = TRIM13 staining intensity vs frequency). Result shown is quantified from 10 000 U937 cells expressing either SCRsh or TRIM13sh2. (B) Representative images of TRIM13 localization (top image, nuclear localized; bottom image, nonnuclear localized) in U937 AML cell line. Scale bar, 7 μm; cell number (top left). (C-E) Pixel analysis of 10 000 cells: (C) U937 T13RINGdel vs T13WT, (D) AML cell lines, (E) CD34⁺ PBSCs and PDXs; TRIM13 localization compared with DNA localization (FxCycle Violet). Higher similarity score means TRIM13 and DNA images are more similar. Nuclear localization indicated by gray shading. (F) Representative images of U937 cells expressing either HA-TRIM13 (top) or HA-NES-TRIM13 (bottom). Scale bar, 7 μm; cell number (top left). (G) Pixel analysis of 10 000 U937 cells expressing HA-TRIM13 or HA-NES-TRIM13. Gray shading indicates nuclear localization. (H) Colony forming unit assay in AML cell lines expressing empty vector, HA-TRIM13, or HA-NES-TRIM13. (I) Pixel analysis of 10 000 CD34⁺ PBSCs or PDXs cells comparing TRIM13 staining intensity to similarity to DNA stain (FxCycle). (J) Pixel analysis of 10 000 CD34⁺ PBSCs or PDXs cells showing the similarity between TRIM13 staining intensity and DNA staining (FxCycle) intensity. ∗P < .05, determined using one-way ANOVA, compared with empty vector (H). Results shown are mean ± SD from 3 biological replicates (H), histogram from 10 000 cells (A,C,D,E,G), or representative of 3 biological replicates (I,J).