Figure 6.
TRIM13 stabilizes proteins associated with cell cycle. (A) Volcano plot comparing identified protein expression from T13WT with T13RINGdel in nuclear or cytoplasmic extracts. Results are calculated from 3 independent submissions. Red shaded region indicates proteins significantly upregulated (P < .05) in T13RINGdel compared with T13WT. (B) Volcano plot comparing differentially ubiquitinated peptides in T13RINGdel vs T13WT U937 cells. Blue shading indicates ubiquitinated peptides significantly lost in T13RINGdel (P < .05). (C) Venn diagram comparing differentially ubiquitinated peptides lost in T13RINGdel with proteins significantly upregulated (P < .05; more than twofold change in peptide spectrum match, or PSMs). (D) Gene ontology (GO) analysis of proteins with a ubiquitination site lost in T13RINGdel U937 cells. (E) Cell cycle (EdU vs FxCycle) analysis in multiple T13RINGdel clones and TRIM13 overexpression on day 5 after infection in each AML cell line. Bars indicate mean ± SD, and ∗P < .05 for each specific cell cycle phase within each cell type compared with CTRLsg, as determined using one-way ANOVA on 3 independent biological replicates. (F) Representative imaging cytometry of CCNA1 and TRIM13 localization through cell cycle in MA9.NRAS. (G) Similarity of CCNA1/TRIM13 through cell cycle. Red dashed line is line of best fit by linear regression. Nonzero slope calculated by linear regression modeling and P value listed. Blue shading indicates more similar TRIM13/CCNA1 than dissimilar, and cell cycle stages listed (G0/G1, S, G2, and M) as determined by DNA content and nuclear morphology. (H) Western blot analysis of PBSC expressing TRIM13sh 72-hours after infection and U937 T13RINGdel AML cell line at steady state. (I) Western blot analysis of T13WT vs T13RINGdel U937 cells expressing a dox-inducible CCNA1 at indicated time points after dox induction. Results shown are representative of 2 biological replicates. (J) Colony assay of indicated genotypes of U937 cells expressing either empty vector or CCNA1 in the presence of dox. Results shown are mean ± SD from 4 biological replicates, dots indicate individual replicates. ∗P < .05 between noted groups (E,H), as determined using one-way ANOVA with Bonferroni post hoc test (H).

TRIM13 stabilizes proteins associated with cell cycle. (A) Volcano plot comparing identified protein expression from T13WT with T13RINGdel in nuclear or cytoplasmic extracts. Results are calculated from 3 independent submissions. Red shaded region indicates proteins significantly upregulated (P < .05) in T13RINGdel compared with T13WT. (B) Volcano plot comparing differentially ubiquitinated peptides in T13RINGdel vs T13WT U937 cells. Blue shading indicates ubiquitinated peptides significantly lost in T13RINGdel (P < .05). (C) Venn diagram comparing differentially ubiquitinated peptides lost in T13RINGdel with proteins significantly upregulated (P < .05; more than twofold change in peptide spectrum match, or PSMs). (D) Gene ontology (GO) analysis of proteins with a ubiquitination site lost in T13RINGdel U937 cells. (E) Cell cycle (EdU vs FxCycle) analysis in multiple T13RINGdel clones and TRIM13 overexpression on day 5 after infection in each AML cell line. Bars indicate mean ± SD, and ∗P < .05 for each specific cell cycle phase within each cell type compared with CTRLsg, as determined using one-way ANOVA on 3 independent biological replicates. (F) Representative imaging cytometry of CCNA1 and TRIM13 localization through cell cycle in MA9.NRAS. (G) Similarity of CCNA1/TRIM13 through cell cycle. Red dashed line is line of best fit by linear regression. Nonzero slope calculated by linear regression modeling and P value listed. Blue shading indicates more similar TRIM13/CCNA1 than dissimilar, and cell cycle stages listed (G0/G1, S, G2, and M) as determined by DNA content and nuclear morphology. (H) Western blot analysis of PBSC expressing TRIM13sh 72-hours after infection and U937 T13RINGdel AML cell line at steady state. (I) Western blot analysis of T13WT vs T13RINGdel U937 cells expressing a dox-inducible CCNA1 at indicated time points after dox induction. Results shown are representative of 2 biological replicates. (J) Colony assay of indicated genotypes of U937 cells expressing either empty vector or CCNA1 in the presence of dox. Results shown are mean ± SD from 4 biological replicates, dots indicate individual replicates. ∗P < .05 between noted groups (E,H), as determined using one-way ANOVA with Bonferroni post hoc test (H).

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