Ab hg fe d c Supplementary Figure 1 Supplementary figure 1: Validation of the ERRα antibody used...
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a b h g f e d c Supplementary Figure 1 Supplementary figure 1: Validation of the ERRα antibody used for immunohistochemical analysis. Control MCF7 breast cancer cell line (a), ERRα siRNA treated MCF7 cells (b). Representative selection of immunohistochemical staining pattern of ERRα; negative nucleus (c), strong nucleus (d), negative cytoplasm (e), strong cytoplasm (f), medium cytoplasm and medium nucleus (g), and a mix of medium and negative nuclei (h). To validate the ERRα antibody used for immunohistochemistry MCF7 cells (ATCC, LGC standards, UK) cultured in phenol-red free Optimem media supplemented with 4% FBS (Gibco by Life Technologies, UK) were transfected with control siRNA (Silencer® Negative Control No. 1; AM4611) or ERRα -directed siRNA (Ambion by Life Technologies, UK; s4829 and s4830) using the Amaxa Nucleofector system (Lonza, Swizerland; Program P-020) with the nucleofection reagent kit V. Transfected cells were harvested 72 h post-transfection, formalin fixed, dehydrated and paraffin
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ER+ Cytoplasmic ERRαNuclear ERRα HR (BCS): 1.46 ( ) HR (RFS): 1.27 ( ) RFS, HR: 0.76 ( ), p=0.31 MFS, HR: 0.81 ( ), p=0.50 BCS, HR: 0.93 ( ), p=0.85 HR (BCS): 0.98 ( ) HR (RFS): 1.18 ( ) RFS, HR: 0.86 ( ), p=0.58 MFS, HR: 0.93 ( ), p=0.82 BCS, HR: 1.32 ( ), p=0.48 Adjuvant untreated Tamoxifen treated A C B D Supplementary Figure 3 Supplementary Figure 3: ERRα protein expression is not prognostic in ER-positive breast cancer. The prognostic value of ERRα levels evaluated according to hormone receptor status in adjuvant untreated patients (A, C), and in tamoxifen-treated patients (B, D). Breast cancer survival (BCS), Recurrence-free survival (RFS), Metastasis-free survival (MFS), Hazard ratio (HR); (95% confidence interval).
Transcript of Ab hg fe d c Supplementary Figure 1 Supplementary figure 1: Validation of the ERRα antibody used...
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Supplementary Figure 1
Supplementary figure 1: Validation of the ERRα antibody used for immunohistochemical analysis. Control MCF7 breast cancer cell line (a), ERRα siRNA treated MCF7 cells (b). Representative selection of immunohistochemical staining pattern of ERRα; negative nucleus (c), strong nucleus (d), negative cytoplasm (e), strong cytoplasm (f), medium cytoplasm and medium nucleus (g), and a mix of medium and negative nuclei (h). To validate the ERRα antibody used for immunohistochemistry MCF7 cells (ATCC, LGC standards, UK) cultured in phenol-red free Optimem media supplemented with 4% FBS (Gibco by Life Technologies, UK) were transfected with control siRNA (Silencer® Negative Control No. 1; AM4611) or ERRα -directed siRNA (Ambion by Life Technologies, UK; s4829 and s4830) using the Amaxa Nucleofector system (Lonza, Swizerland; Program P-020) with the nucleofection reagent kit V. Transfected cells were harvested 72 h post-transfection, formalin fixed, dehydrated and paraffin embedded. Sections of 4µm were prepared for immunohistological staining according to the protocol used for TMAs.
ER- Cytoplasmic ERRα Nuclear ERRα
A B
0 5 10 15 20 25 30
Years
0.0
0.2
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1.0
BC
S
Low (n=21)
High (n=66)
p=0.490 5 10 15 20 25 30
Years
0.0
0.2
0.4
0.6
0.8
1.0
BC
S
Low (n=47)
High (n=40)
p=0.015
HR (BCS): 1.46 (0.49-4.32) HR (RFS): 1.08 (0.47-2.49)
HR (BCS): 2.94 (1.19-7.21) HR (RFS): 1.73 (0.86-3.48)
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Supplementary Figure 2
Supplementary Figure 2: The prognostic value of ERRα protein expression and subcellular location in adjuvant-untreated ER-negative patients. The prognostic value of ERRα levels evaluated according to hormone receptor status in adjuvant untreated patients (A-B). Breast cancer survival (BCS), Hazard ratio (HR); (95% confidence interval).
ER+ Cytoplasmic ERRα Nuclear ERRα
0 5 10 15 20 25 30
Years
0.0
0.2
0.4
0.6
0.8
1.0
BC
S
Low (n=146)
High (n=144)
p=0.76
0 5 10 15 20 25 30
Years
0.0
0.2
0.4
0.6
0.8
1.0
BC
S
Low (n=149)
High (n=47)
p=0.95
0 5 10 15 20 25 30
Years
0.0
0.2
0.4
0.6
0.8
1.0
RFS
High (n=148)
Low (n=171)
p=0.58
0 5 10 15 20 25 30
Years
0.0
0.2
0.4
0.6
0.8
1.0
RFS
High (n=172)
Low (n=147)
p=0.31
HR (BCS): 1.46 (0.49-4.32)HR (RFS): 1.27 (0.83-1.93)
RFS, HR: 0.76 (0.45-1.29), p=0.31MFS, HR: 0.81 (0.43-1.51), p=0.50BCS, HR: 0.93 (0.44-1.98), p=0.85
HR (BCS): 0.98 (0.58-1.68)HR (RFS): 1.18 (0.77-1.79)
RFS, HR: 0.86 (0.50-1.47), p=0.58MFS, HR: 0.93 (0.49-1.75), p=0.82BCS, HR: 1.32 (0.62-2.80), p=0.48
Adj
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Supplementary Figure 3
Supplementary Figure 3: ERRα protein expression is not prognostic in ER-positive breast cancer. The prognostic value of ERRα levels evaluated according to hormone receptor status in adjuvant untreated patients (A, C), and in tamoxifen-treated patients (B, D). Breast cancer survival (BCS), Recurrence-free survival (RFS), Metastasis-free survival (MFS), Hazard ratio (HR); (95% confidence interval).
