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. 2019 Apr 18;8(5):30. doi: 10.1038/s41389-019-0139-x

Fig. 3. TRIM56 depletion decreases ER alpha signaling activity in breast cancer cells.

Fig. 3

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 ERα 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 ERα 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 ERα 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