TRIM59 promotes breast cancer cell proliferation, growth, migration, and invasion in vitro.

(A) IB analysis of TRIM59 expression in the noncancerous mammary epithelial cell line (MCF10A) and breast cancer cell lines (MCF7 and MDA-MB-231). (B and C) IB analysis of TRIM59 expression in shRNA or CRISPR/Cas9 lentivirus-transduced control, TRIM59 KD (shTRIM59-2, KD efficiency confirmed by qPCR; B, middle) and TRIM59 KO MCF7 (B, left), or TRIM59-overexpressing (OE) MDA-MB-231 stable cells (C). sgRNA targeting TRIM59 was confirmed by Sanger sequencing (B, right). A dinucleotide GT insertion (underlined) caused an early abortion of TRIM59 translation, as further validated by immunoblotting (B, lane 4). (D and E) Graphic representation of 96-hour MTS proliferation assays of WT (non-transduced), control (transduced with non-targeting shRNA/sgRNA), shTRIM59 and TRIM59 KO MCF7 cells (D), and WT or TRIM59 OE MDA-MB-231 cells (E). (F and G) Colony formation assay in 3D Matrigel culture was performed to assess the rate of colony formation of control, shTRIM59, and TRIM59 KO MCF7 cells at day 8 (F) or WT and TRIM59 OE MDA-MB-231 cells at day 4 (G). Bar graphs show the number of colony-forming units (n = 8). Scale bars: 100 μm. (H and I) Scratched wound healing assays were performed to assess the rates of migration for control, shTRIM59, and TRIM59 KO MCF7 cells at 72 hours (H) or WT and TRIM59 OE MDA-MB-231 cells at 36 hours (I). Bar graphs show the percentage of migration rate (n = 8). Scale bars: 100 μm. (J and K) Transwell invasion assay was performed in Matrigel-coated 8-μm pore size transwell plates to assess the invasion ability of control, shTRIM59, and TRIM59 KO MCF7 cells (J) or WT and TRIM59 OE MDA-MB-231 cells (K). Bar graphs show the numbers of invaded cells (n = 8). Scale bars: 100 μm. Data in B, D-K are presented as means ± SD; *P P S1 Data. CRISPR/Cas9, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease; IB, immunoblot; KD, knockdown; KO, knockout; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; OD, optical density; OE, overexpressing; qPCR, quantitative polymerase chain reaction; sgRNA, single guide RNA; shRNA, short hairpin RNA; TRIM59, tripartite motif 59; WT, wild-type.