Table 1.
Details of antibody and dilution.
Figure 1.
Immunohistochemical (IHC) staining of NgBR, Nogo-B and survivin in non-neoplastic breast epithelium and invasive ductal carcinoma (IDC).
Staining was developed using NovaRed as described in methods. Images were taken using an Olympus microscope with x20 lens. (A–C) IHC staining of NgBR, Nogo-B and survivin in normal breast tissues. Few epithelial cells are positive for NgBR (A) and Nogo-B (B), and the majority of myoepithelial cells express NgBR (A) and Nogo-B (B). Few epithelial and myoepithelial cells are weak positive for survivin (C). (D–F) IHC staining of NgBR, Nogo-B and survivin in luminal A IDC with strongly positive staining of NgBR (D), Nogo-B (E) and survivin (F). The positive staining was localized in the cytoplasm of tumor cells with few tumor cells demonstrating membrane staining of Nogo-B and survivin. (G–I) IHC staining of NgBR, Nogo-B and survivin triple negative IDC with negative staining of NgBR (G), Nogo-B (H) and survivin (I) in all tumor cells.
Table 2.
Protein expression in invasive ductal carcinoma (N = 400).
Table 3.
Correlation analysis of NgBR and survivin in different stages of breast invasive ductal adenocarcinoma.
Figure 2.
NgBR and survivin transcripts in breast tumor tissues determined by real-time PCR.
Normalized human breast tumor qPCR panels were utilized (Origene). The copy number of NgBR and survivin was determined by real-time PCR. (A) NgBR RNA levels in ductal adenocarcinoma specimens were presented as fold changes as compared to the average NgBR RNA levels of all normal breast tissue (* tumor vs normal, p<0.05). (B) Survivin RNA levels in ductal adenocarcinoma specimens were presented as fold changes as compared to the average survivin RNA levels of all normal breast tissue (* tumor vs normal, p<0.05).
Table 4.
Correlation analysis of NgBR and survivin transcripts in different stages of ductal adenocarcinoma.
Figure 3.
NgBR regulates estradiol-induced survivin expression in estrogen receptor positive breast tumor cells.
T47D is an estrogen receptor positive breast tumor cell line. MDA-MB-468 is an estrogen receptor negative breast tumor cell line. NgBR was knockdown in both T47D and MDA-MB-468 cells using siRNA as described in methods. Both tumor cells were treated with 10 nM estradiol for 48 hours. Protein levels of NgBR, ER-alpha and survivin were determined by Western blot analysis. Beta-Actin is applied as a housekeeping protein. The density of each band was measured using NIH ImageJ and presented as relative intensity of survivin after normalized with beta-actin housekeeping protein. (A) NgBR knockdown diminished estradiol-induced survivin expression in T47D breast tumor cells. (B) Quantitative analysis of survivin protein level change in T47D cells by measuring intensity of survivin western blot band. Data is presented as fold changes of estradiol treatment group as compared to the non-treatment group (n = 3; * siNgBR vs NS p<0.05). (C) NgBR knockdown has no effect on survivin expression in MDA-MB-468 breast tumor cells. (D) Quantitative analysis of survivin protein level change in MDA-MB-468 cells by measuring intensity of survivin western blot band. Data is presented as fold changes of estradiol treatment group as compared to the non-treatment group. (n = 3; siNgBR vs NS p = 0.092).
Figure 4.
NgBR is essential for estradiol-induced survivin expression and cell growth of MCF-7 breast tumor cells.
MCF-7 is an estrogen-dependent breast cancer cell line. NgBR was knocked down in MCF-7 cells using siRNA. (A) NgBR knockdown diminished estradiol-induced survivin expression in MCF-7 breast tumor cells. MCF-7 cells were treated with 10 nM estradiol for 6 and 48 hours. Protein levels of NgBR and survivin were determined by Western blot analysis. Beta-Actin is applied as a housekeeping protein. The density of each band was measured using NIH ImageJ and presented as relative intensity of survivin after normalized with beta-actin housekeeping protein. (B) Folds of survivin increase were determined by measuring relative western blot intensity of survivin. Data is presented as fold changes of estradiol treatment group as compared to the non-treatment group (n = 3; 6 hrs estradiol treatment, NS vs siNgBR p = 0.178; * 48 hrs estradiol treatment, NS vs siNgBR p<0.05). (C) NgBR knockdown impaired estradiol-stimulated cell growth of MCF-7 breast tumor cells. MCF-7 cells were treated with 10 nM estradiol for 24 and 48 hours. Viable cell numbers were counted using the Bio-Rad TC10™ Automated Cell Counter. Data is presented as mean±SEM (n = 3, # 24 hrs or 48 hrs estradiol treatment vs baseline p<0.05; * siNgBR vs NS p<0.05); E2: estradiol.