MCF-7 and HEK293 cells were ordered form American Type Culture Collection (ATCC). MCF-7 and HEK293 cells were cultured in Dulbecco’s Modified Eagle’s Medium that contains 4,5 g/L glucose and 4 mM L-glutamine (DMEM, 41965, Life Technologies) supplemented with 10% Fetal Bovine Serum (FBS, 10270, Life Technologies). All cell lines were subject to cell line authentication. The cell line authentication via Short Tandem Repeat (STR) was performed via PowerPlex 21 system. The STR data of HEK293 and MCF-7 lines were found consistent with STR data in ATCC.
Plasmids and siRNA
The Flag-TRIM-3 plasmid was acquired from Origene. The HA-K48 and Ub wild type plasmids were acquired from our previous study . The P53 plasmid was acquired form previous studies . The TRIM3 and P53 deletion variants were sub-cloned from the original plasmids. The Lipofectamin 2000 (1662298, Invitrogen) was used for the plasmids transfection. Small interfering RNAs were used for specific gene knocking-down. The TRIM3 siRNA sequences were: CAAACGAAAGGACAACCCAdTdT; UGGGUUGUCCUUUCGUUUGdTdT and GCAACAACCAGUGUAUUCAdTdT; UGAAUACACUGGUUGUUGCdTdT. The negative control siRNA sequences were: UUCUCCGAACGUGUCACGUTT; ACGUGACACGUUCGGAGAATT. The RNAiMAX reagent (13778150, invitrogen) was used for siRNA transfection.
RNA extraction and qPCR analysis
RNeasy plus mini kits were used to extract total RNA (Qiagen). Real-time PCR was performed as previously described . 36B4 was used for internal control. The primer sequences were shown here. 36B4: F: GGCGACCTGGAAGTCCAACT; R: CCATCAGCACCACAGCCTTC. P21: F: GTGGCTCTGATTGGCTTTCTG; R: CTGAAAACAGGCAGCCCAAG. BTG2: F: AGACGAGGCAAAGCGGTAAA; R: TCCAACCATTCACGGTCAGA. P53INP1: F: TATGCTGCCCATTTCATTT; R: CTGTGCATAACTCCTGCCCT.
Quantification of cell viability
MCF-7 cells were transfected with siTRIM3 or siControl into 24-well plates. Twenty-Four hours after transfection, the cells number was countered and 4000 cells were seeded into 96-well plates. The relative cell viability was measured at indicated time points. Cell numbers were determined using the WST-1 cell proliferation reagent as previously described.
EdU staining assay
For ethynly-deoxyuridine (EdU) labeled DNA, cells were incubated with EdU for 2 hours. Later on, the cells were fixed in cell culture plates with 4% formalin. The EdU positive cells were counted with statistical analysis.
Flow cytometry assay
For the cell cycle analysis, the MCF-7 cells were transfected with 50 uM siTRIM3 or siControl. After 24 hours, cells were fixed via 70% ethanol and stained with propidium iodide. For the apoptosis assay, the MDAMB175 cells were transfected with 50 uM siTRIM3 or siControl. Twenty-four hours post-transfection, cell were treated with 10 uM cisplatin for 8 hours. Aftern 24 hours, cells were stained with propidium iodide and annexin V. The BD LSR flow was used to measure the fluorescence intensity.
Cells were harvested and lysed with RIPA buffer. Proteins were separated by electrophoresis on SDS-polyacrylamide gel electrophoresis (PAGE) and electro-transferred to PVDF membrane. The antibodies used in this study were listed here: Anti-TRIM3 (HAP043396, Sigma); Anti-P53 (SC-126, Santa Cruz); Anti-Cleaved Caspase-3 (ab2302, Abcam); Anti-HA (MMS-101R, COVANCE); Anti-myc (9E10, ab32, Abcam); Anti-myc (Ab9106, Abcam); Anti-Actin (GB12001, Servicebio). Membranes were then washed with PBS for three times and incubated with secondary antibodies Peroxidase-Conjugated AffiniPure Goat Anti-Mouse IgG or Goat Anti-Rabbit IgG. Fluorescent signals were visualized with ECL system. (amersham imager 600, USA).
Immunoprecipitation was performed as described in previous study . The MCF-7 cells total cell lysis was pre-cleared with rabbit IgG for 2 h and subsequently immunoprecipitated with TRIM3 antibody (HAP043396, Sigma) over night, while rabbit IgG (Santa Cruz) was used as the negative control. The bounded protein was analyzed by Anti-P53 antibody (SC-126, Santa Cruz). For the domain CoIP assay, the P53 or TRIM3 variants were transfected into HEK293 cells. The total cell lysis was pre-cleared with rabbit IgG for 2 hours and subsequently immunoprecipitated with Myc antibody overnight, while the rabbit IgG was used as the negative control. The bounded protein was analyzed via GFP antibody (Ab290, Abcam).
Protein stability assays
About 105 MCF-7 cells were seeded into twenty-four well plates and transfected with 50 uM TRIM3 siRNA or siControl. After 48 h, cells were treated with 100uM cycloheximide (C7698, Sigma) for indicated time points. Samples were subject to western blot for P53 degradation.
Poly-ubiquitination detection assay
To directly detect the enriched K48-ubiquitinated and total ubiquitinatoin P53 from the cell extracts, HEK293 cells were transfected with 0.8 ug K48 Ubi or 0.8 ug Ub plasmids together with 0.8 ug GFP-P53 plasmid and 0.8 ug Myc-TRIM3 or Myc-vector. After 24 h, the cells were treated with 20 uM MG132 for 7 hours,then total protein was extracted and pre-cleared with 30ul protein A (santa cruz, SC-2001) for 4 h. The supernatant was collected and immunoprecipitated by P53 antibody. Western blot with HA antibody was performed to detect total or K48 poly-ubiquitinated P53.
MCF-7 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit anti-TRIM3 polyclonal antibody (HAP043396, Sigma) and mouse anti-P53 monoclonal antibodies (SC-126, Santa Cruz) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using Nikon A+ laser scanning confocal system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using ImageJ.
Student's t-test, Pearson correlation coefficient, and Cox regression analysis were used for comparisons. A P-value of < 0.05 was considered to be significant.