Fig 2: TRIM56 relates to poor endocrine treatment outcome and correlates with ER alpha and PR protein levels in human breast cancer samples.A–C TRIM56 mRNA level correlates with poor endocrine treatment outcome in breast cancer patients. Each panel is derived from independent clinical cohorts. These clinical data are acquired from KMPLOT database (http://kmplot.com/analysis/). D Examples of positive/negative TRIM56, ERa, PR and HER2 staining in breast tumor samples were shown by ×100 magnification. The statistical data of each protein marker are shown in Table 1

Fig 3: TRIM56 depletion decreases ER alpha signaling activity in breast cancer cells.A Top ten signaling pathways significantly decreased/increased by TRIM56 depletion in MCF7 cells. The pathway-enrichment analysis was used by the threshold P < 0.001 and fold change >2 to derive regulated genes. TRIM56 was depleted by siRNA (mix of siTRIM56 #1 and siTRIM56 #2) or treated with siControl. After 48 h, the whole mRNA was extracted for RNA sequence analysis. The siControl and siTRIM56 were done in triplicates. B The heat-map graph shows the ERa regulating genes, which is significantly inhibited by TRIM56 depletion in MCF-7 cells. The pathway-enrichment analysis was used by the threshold P < 0.001 and fold change >2 to derive regulated genes. TRIM56 was depleted by siRNA (mix of siTRIM56 #1 and siTRIM56 #2) or treated with siControl. After 48 h, the whole mRNA was extracted for RNA sequence analysis. The siControl and siTRIM56 were done in triplicates. C TRIM56 depletion effect by two different siRNA oligos. MCF-7 cells are transfected with two independent TRIM56 siRNAs or siControl. After 48 h, TRIM56 mRNA levels are determined by QPCR. 36B4 was used as internal control. *P < 0.05; **P < 0.01; ***P < 0.001 for TRIM56 mRNA level comparison. D TRIM56 depletion effect on ER alpha protein level by two different siRNA oligos. MCF-7 cells were transfected with two independent TRIM56 siRNAs or siControl. TRIM56 and ER alpha protein levels were determined by the western blot analysis. Actin was used as internal control. E TRIM56 depletion decreases ERa target genes using two different siRNA oligos. MCF-7 cells were transfected with siTRIM56 or siControl. After 48 h, total RNA was prepared and the expression of the endogenous ERa target genes, PS2, GREB1, and PDZK1 were determined by qPCR. Shown are the results from three experiments. *P < 0.05; **P < 0.01; ***P < 0.001 for target gene expression comparison. F TRIM56 depletion effect on ER alpha protein level. MCF-7 cells were transfected with siTRIM56 or siControl. After 48 h, cells were treated with either ethanol or 10 nM estradiol for 6 h. TRIM56 and ER alpha protein levels were determined by the western blot analysis. Actin was used as internal control. G TRIM56 depletion decreases ER alpha target genes. MCF-7 cells were transfected with siTRIM56 or siControl. After 48 h, cells were treated with either ethanol or 10 nM estradiol for 6 h. Total RNA was prepared and the expression of the endogenous ER alpha target genes, PS2, GREB1, and PDZK1 were determined by qPCR. Shown are the results from three experiments. *P < 0.05; **P < 0.01; ***P < 0.001 for target gene expression comparison. H TRIM56 depletion affects ERE-luciferase activity in MCF-7 cells. MCF-7 cells were transfected with siTRIM56 or siControl together with ERE luciferase reporter plasmid. Cells were treated with 10 nM estradiol or vehicle. Luciferase activity was measured 48 h after transfection. Shown are the results from three experiments. *P < 0.05; **P < 0.01; ***P < 0.001 for luciferase activity comparison

Fig 4: TRIM56 associates with ER alpha and increases its stability.A Intracellular localization analysis of TRIM56 and ER alpha by immunofluorescence assay. MCF7 cells were cultured in normal medium before fixation. Intracellular localization of TRIM56 (green) and ER alpha (red) was shown. Nuclei (blue) were stained with 4′,6-diamidino-2-phenylindole (DAPI). B TRIM56 protein could not shuttle into the nucleus by estradiol treatment. Cells were subject to estradiol treatment for 30 min. The subcellular protein fractionation kit (Thermo Scientific, 78840) was used for cytoplasm and nuclear separation. Tubulin and Histone-3 were used for cytoplasm and nuclear control. C Co-IP assay reveals association between endogenous TRIM56 and ER alpha in MCF7 cells. MCF-7 cells were harvested with RIPA lysis buffer. Co-IP was performed using antibody as indicated. D Pulldown assay reveals direct interaction between TRIM56 and ER alpha. ER alpha recombinant protein was incubated with GST-TRIM56 or GST protein. The interacted ER alpha signaling was detected via western blot. E TRIM56 is mainly localized in the cytoplasm and associates with ER alpha in the cytosol. The subcellular protein fractionation kit (Thermo Scientific, 78840) was used for cytoplasm and nuclear separation. Tubulin and Histone-3 were used for cytoplasm and nuclear control. Based on the separation, IP was done by TRIM56 antibody in both the cytosol and nuclear lysis. ER alpha antibody was used to detect the interaction in both the cytosol and nuclear. F TRIM56 has stabilization effect on ER alpha in HEK293 cells. HEK293 cells were transfected with 2 µg ERα plasmid and 0.5 µg Myc-tag or Myc-TRIM56 plasmids. After 24 h, cell lysates were prepared for western blot analysis. The results are representative for three independent experiments. G In the presence of the proteasome inhibitor MG132, the stabilization effect of TRIM56 on ER alpha did not further increase ER alpha protein levels. HEK293 cells were transfected with 2 µg ERα plasmid and 0.5 µg Myc-tag or Myc-TRIM56 plasmids. After 24 h, cells were treated with 10 µM MG132/vehicle for 6 h. Cell lysates were prepared for western blot analysis. The results are representative for three independent experiments. H TRIM56 increases ER alpha half-life in HEK293 cells. HEK293 cells were transfected with HA-ERα plasmid and Myc-tag or Myc-TRIM56 plasmids. After 24 h, cells were treated with 100 µM cycloheximide/vehicle for indicated times. Cell lysates were prepared for western blot analysis. The results are representative for three independent experiments. The ER alpha relative density was measured by ImageJ software. I TRIM56 depletion decreases ER alpha half-life in MCF-7 cells. MCF-7 cells were transfected with siTRIM56 or siControl. After 24 h, cells were treated with 100 µM cycloheximide/vehicle for indicated times. Cell lysates were prepared for western blot analysis. The results are representative for three independent experiments. The ER alpha relative density was measured by ImageJ software

Fig 5: TRIM56 depletion inhibits ER-alpha-positive breast cancer cell proliferation in vitro and in vivo.A TRIM56 depletion inhibits the cell proliferation in breast cancer cells. MCF-7 cells were transfected with 50 nM TRIM56 siRNA (mix of #1 and #2) or 50 nM control siRNA. After 24 h, the WST assay was used to determine the cellular metabolic activity at indicated time points after transfection. Experiments were done in triplicates. *P < 0.05; **P < 0.01; ***P < 0.001 for cell growth comparison. B TRIM56 depletion inhibits estrogen-driven cell proliferation in breast cancer cells. MCF-7 cells were transfected with 50 nM TRIM56 siRNA (mix of #1 and #2) or 50 nM control siRNA. After 24 h, cells were treated with vehicle or 10 nM estradiol. The WST assay was used to determine the cellular metabolic activity at indicated time points after transfection. Experiments were done in triplicates. *P < 0.05; **P < 0.01; ***P < 0.001 for cell growth comparison. C TRIM56 depletion significantly induces G1 cell cycle arrest in breast cancer cells. MCF-7 cells were transfected with 50 nM TRIM56 siRNA (mix of #1 and #2) or 50 nM control siRNA. After 48 h, cells were harvested and fixed by 70% ethanol. The cell cycle phase was anaylzed by PI staining. Experiments were done in triplicates. *P < 0.05; **P < 0.01; ***P < 0.001 for cell growth comparison. D Clone formation assay of MCF-7 cells transfected with indicated 50 nM TRIM56 siRNA (mix of #1 and #2) or 50 nM control siRNA. Quantification of clone formation is shown at the indicated time points. Data are presented as ±SD. **P < 0.01, ***P < 0.001 (Student’s t test). E Wound-healing assay of MCF-7 were transfected with indicated 50 nM TRIM56 siRNA (mix of #1 and #2) or 50 nM control siRNA. Quantification of wound closure at the indicated time points. Data are presented as ±SD. **P < 0.01, ***P < 0.001 (Student’s t test). F TRIM56 depletion promotes apoptotic signaling in breast cancer cells. T47D cells were transfected with indicated 50 nM TRIM56 siRNA (mix of #1 and #2) or 50 nM control siRNA. After 24 h, cells were harvested for western blot analysis. The cleaved caspase-3 was detected to indicate apoptosis signaling activity. G TRIM56 depletion inhibits the cell proliferation in breast cancer cells in vivo. MCF-7 cells were stably transfected with lentivirus carrying a scrambled shRNA or TRIM56 shRNA. Female NOD scid gamma (NSG) mice were estrogen-supplemented by implantation of slow-release 17ß-estradiol pellets (0.72 mg/90-d release; Innovative Research of America) 1 day before MCF-7 tumor cell injection into the mammary fat pad (2 × 106 MCF-7 cells suspended in 100 µl Matrigel solution). MCF-7 tumor xenografts were measured every 10 days and the tumor volume was calculated by length × width2 /2. The mice were killed 2 months after transplant. The tumor growth curve and a photograph were shown