Cell culture

The human breast cancer cell lines Hs578Bst, MCF7, MDA-MB-453, T-47D, ZR-75-1, ZR-75-30, BT-20, BT-549, CAMA-1, HCC1806, HCC1937, Hs578T, MDA-MB-231, and MDA-MB-468 were cultured according to the manufacturer’s instruction. Short tandem repeat (STR) profiling were used and authenticated in all cell lines.

Chemical reagents

Olaparib was purchased from https://www.selleck.cn/ (AZD2281).

Tissue specimens and immunohistochemistry

This study was conducted on a total of 39 paraffin-embedded TNBC samples. 39 cases of paraffin-embedded TNBC was used to detect the expression of TRIM47 using Anti-TRIM47 antibody (ab72234) and Anti-BRCA1 antibody [MS110] (ab16780). For the use of these clinical materials for research purposes, prior patient consent and approval from the Institutional Research Ethics Committee were obtained. The 39 specimens were histopathologically and clinically diagnosed in Integrated Hospital of Traditional Chinese Medicine, Southern Medical University. 10 freshly TNBC tissues were histopathologically and clinically diagnosed in the First Affiliated Hospital of Sun Yat-sen University were frozen and stored in liquid nitrogen until further use.

Vectors, retroviral infection, and transfection

pMSCV/TRIM47 recombinant plasmid was generated by subcloning the PCR-amplified human TRIM47 coding sequence into pMSCV vector. pMSCV/BRCA1 recombinant plasmid was generated by subcloning the PCR-amplified human BRCA1 coding sequence into pMSCV vector. TRIM47 ring-domain mutant (C9S/C12S) plasmid was generated by using QuikChange ® Site-Directed Mutagenesis Kit according to manufacturer’s instructions. To silence endogenous TRIM47, two siRNA oligonucleotides were cloned to generate pSuper-retro-shTRIM47#1, shTRIM47#2, respectively. The primers using in this study were listed in Supplementary Table 1. Transfection of siRNA or plasmids was performed using the Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. Stable cell lines expressing TRIM47 or shTRIM47#1, shTRIM47#2 were selected for 10 days with 0.5 μg/ml puromycin 48 h after infection.

Immunoprecipitation analysis

Immunoprecipitation assay was performed according to described previously [37]. Lysates were incubated with Flag or His affinity beads (Sigma-Aldrich). The agarose beads were washed with wash buffer. Then the elutions were detected using appropriate antibodies.

Far western blotting

The indicated proteins were immunoprecipitated by Flag-tag affinity gel (Sigma-Aldrich, St. Louis, MO) and resolved by SDS-PAGE, and the proteins were transferred to PVDF membrane. The membrane was then blocked in 10% skimmed milk for 1 h at 4 °C. As indicated, recombinant His-TRIM47 or Flag-BRCA1 was added at 1 μg/ml and incubated at 4 °C for 18 h. The blot was then washed 6 times with TBST and subjected to WB analysis using anti-His or anti-Flag antibody (Sigma-Aldrich, St. Louis, MO).

Western blotting analysis

Western blotting was performed using antibodies against Anti-TRIM47 antibody (ab72234), Anti-p53 antibody [PAb 240] (ab26), Anti-p21 antibody [EPR362] (ab109520), Anti-p27 KIP 1 antibody [Y236] (ab32034), Anti-rH2AX antibody [SIGMA] (H5912), Anti-BARD1 antibody [2059C4a] (ab50984), Anti-Rad51 antibody [EPR4030(3)] (ab133534), Anti-GFP antibody (ab290), and Anti-BRCA1 antibody [MS110] (ab16780). The membranes were re-probed with an Anti-GAPDH antibody [6C5] - Loading Control (ab8245) as the loading control.

Quantitative real-time reverse transcription PCR (qPCR)

Total RNA was isolated with TRIzol reagent (Invitrogen) according to manufacturer’s instructions. RNA was reverse-transcribed into cDNA and carried out via Real-time PCR with SYBR Green Master (Roche). The data were assessed based on the threshold cycle (Ct), and calculated as \(^\,}\,})}\,-\,\,}\,})}]}\), which was normalized to GAPDH expression. All primers are listed as Supplementary Table S1.

Immunofluorescence (IF) staining

IF staining was carried out on indicated cells grown on coverslips using Anti-Rad51 antibody [EPR4030(3)] (ab133534), Anti-rH2AX antibody [SIGMA] (H5912), and FITC-conjugated goat anti-mouse secondary antibody (Jackson Immuno Research). The images were captured using the AxioVision Rel.4.6 computerized image analysis system (Carl Zeiss).

Colony formation assay

Colony formation assay was performed according to described previously [38]. Indicated cells were plated on 60 mm plates (0.5 × 103 cells per plate) and incubated at a level of 5% CO2 at a temperature of 37 °C for 2 weeks. The colonies were stained with 1.0% crystal violet for 30 s after fixation with 10% formaldehyde for 5 min. Then the cells were counted and analyzed.

Luciferase activity assay

Luciferase activity assay was performed according to described previously [38]. Luciferase reporter plasmid and pRL-TK Renilla plasmid were transfected into indicated cells. After 48 h, the cells were lysis and measured using a Dual Luciferase Reporter Assay (Promega) according to the manufacturer’s instructions.

Annexin-V assay

Annexin-V assay was performed according to described previously [39]. For evaluation of apoptosis, PE Annexin V Apoptosis Detection Kit I (BD Pharmingen) was used. Briefly, 1 × 106 indicated treated cells were collected and washed with PBS and the Annexin-V binding solution, subsequently added 150 μl of an Annexin-V antibody in Binding Buffer and incubated for 15 min, followed by addition of 1.5 μl of PI at 1 mg/ml and a further incubation for 5 min at room temperature in the dark. Cell morphology was assessed by phase-contrast microscopy. Percentage of apoptosis was analyzed with an EPICS XL flow cytometer (Beckman-Coulter). Each sample was analyzed in triplicate.

Tumor xenografts

Six-week-old BALB/c-nu mice were purchased from the Center of Experimental Animal of Guangdong Province (Guangdong, China), and approval for the experiments was obtained from the Institutional Animal Care and Use Committee.

To form tumor xenografts in female six-week-old BALB/c nude mice, Vector or TRIM47-transfected-MDA-MB-231 cells (1 × 107) were injected subcutaneously in the back under aseptic conditions with or without Olaparib. Tumor size was measured using callipers every week, and tumor volume was calculated according to the following equation: (long axis × short axis2)/2. The xenograft tumors were removed on the sixth week after the injection and weighed after dissection.

Data processing and visualization

For the relationship between TRIM47 and the OS, RFS, and MFS of breast cancers, Kaplan–Meier Plotter ((http://kmplot.com/analysis) was used. The datasets are available in The Cancer Genome Atlas (TCGA) (https://tcga-data.nci.nih.gov/tcga/). Gene set enrichment analysis (GSEA) was performed on GSEA 2.0.9 (http://www.broadinstitute.org/gsea/).

Statistics

SPSS 21.0 statistical software (IBM, Armonk, NY, USA) was used to analyze the data. Differences between two variables were evaluated using Student’s t-test. The relationship between TRIM47 expression and the clinicopathological characteristics were carried out using the chi-squared test, log-rank analysis, the Kaplan–Meier method, and the Cox regression model. P < 0.05 was considered statistically significant.