Fig 3. TRIM59 promotes the tumor growth and survival of breast cancer cells in vivo.

(A and B) Representative tumor images (A) and tumor volume at indicated time points (B) in nude mice that were orthotopically injected into mammary fat-pad with control (non-targeting shRNA) or shTRIM59 MCF7 cells (1 × 10⁷ cells/mouse). n = 9 mice per group. (C and D) Representative tumor images (C) and tumor size quantification at indicated time points (D) in NSG mice that were orthotopically injected into mammary fat-pad with control and TRIM59 OE MDA-MB-231 cells (5 × 10⁶ cells/mouse). n = 5 mice per group. (E and F) Representative image of shControl or shTRIM59 MCF7 xenografts (E) and control or TRIM59 OE MDA-MB-231 (F) xenograft tumors stained with HE and Ki-67. Scale bars: 20 μm. (G) Cell cycle analysis on Control, shTRIM59, and TRIM59 KO MCF7 cells by flow cytometry. The population of cells at each stage was shown in the bar graph. n = 6. **P < 0.01 versus control. (H and I) Apoptotic assessment of shControl, shTRIM59, and TRIM59 KO MCF7 cells. Apoptosis was assayed with Annexin V/7-AAD staining and measured by flow cytometry (H). Statistical analysis of cell populations at early or late stages of apoptosis (I). Live, 7’AAD⁻AnnexinV⁻; Early, 7’AAD⁻AnnexinV⁺; Late, 7’AAD⁺AnnexinV⁺. n = 6. (J) Representative images of cleaved-caspase3 expression in paraffin sections from xenograft tumors made of shControl or shTRIM59 MCF7 cells (top) and WT or TRIM59 OE MDA-MB-231 cells (bottom). n = 6. Scale bars: 10 μm. (K) IB analysis of cell cycle/apoptosis regulators (p53 and MAP kinase pathway members) expression in shControl or shTRIM59 MCF7 cells. Data in B, D, G, and I are presented as means ± SD. *P < 0.05, **P < 0.01 versus controls. The underlying data can be found in S1 Data. Casp3, caspase3; HE, hematoxylin–eosin; Ki-67, marker of proliferation; KO, knockout; MAP, mitogen-activated protein; NSG, NOD scid gamma; OE, overexpressed; p-ERK, phosphorylation of extracellular signal–regulated kinase (ERK); p-JNK, phosphorylation of c-Jun N-terminal kinase (JNK); p-P38, phosphorylation of P38 MAP kinase; p53, tumor protein 53; shRNA, short hairpin RNA; TRIM59, tripartite motif 59; WT, wild-type.