PMC

Kaplan–Meier analysis of overall survival for ERO1-α expression in 56 cases of invasive TNBC.   

Expression of ERO1-α in TNBC was involved in angiogenesis in clinical cases. (A) TNBCs were classified into four groups by stainability for ERO1-α. (B) The correlations of TNBCs with expression of ERO1-α and numbers of blood vessels were examined. The P-values are given for differences in median values. *P<0.05, **P<0.001, Tukey's test.

Expression of ERO1-α in TNBC tissue was enhanced compared with that in normal breast tissue. (A) Human ERO1-α mRNA levels in TNBC cell lines, TNBC tissues and normal breast tissue determined by real-time PCR. (B) Western blot analysis of TNBC cell lines, TNBC tissue and normal breast tissue. (C) Normal breast tissue and (D) TNBC tissue were stained for ERO1-α ( × 100, inset: × 200). *P<0.05, unpaired Student's t-test.

Expression of ERO1-α promoted tumour growth via augmenting angiogenesis. (A) Western blot analysis of MDA-MB-231 (wild-type (WT)) cells, scrambled shRNA-transfected (SCR) cells and ERO1-α knockdown (sh221 and sh222) cells. (B) Tumour growth rates of SCR, sh221 and sh222 cells were compared in NOD/SCID mice (five animals per group). (C) The numbers of blood vessels within SCR and KD cells per high-power field were compared. (D) Western blot analysis of mock cells and ERO1-α-overexpressed (9A3, 9C1 and 9C2) cells. (E) Tumour growth rates of mock, 9A3, 9C1 and 9C2 cells were compared in NOD/SCID mice (five animals per group). (F) The numbers of blood vessels within mock cells and OE cells per high-power field were compared. ^#P<0.001, Mann–Whitney's U-test. *P<0.01, **P<0.001, unpaired Student's t-test.

ERO1-α influenced the VEGF secretion via both promoting disulfide formation and the mRNA expression level. (A) Concentrations of VEGF in the 24-h culture supernatants from SCR, sh221 and sh222 cells were measured using ELISA. (B) VEGF mRNA expression levels in SCR, sh221 and sh222 cells were determined by real-time PCR analysis. (C, D) Redox status of VEGF in SCR and KD cells was examined by Western blotting under reducing (R) or non-reducing (NR) conditions. Reduced form (R) and oxidised form (O) of VEGF are indicated. The ratio of the oxidised form (O) and reduced form (R) obtained in SCR cells was set as 1, and the differences in the ratio of the oxidised form (O) and reduced form (R) induced by sh221 cells were plotted. (E) Concentrations of VEGF in the 24-h culture supernatants from non-treated MDA-MB-231 cells and MDA-MB-231 cells treated with an ERO1-α inhibitor were measured using ELISA. (F) VEGF mRNA expression levels in non-treated MDA-MB-231 cells and MDA-MB-231 cells treated with an ERO1-α inhibitor were determined by real-time PCR analysis. (G) Concentrations of VEGF in the 24-h culture supernatants from mock, 9A3, 9C1 and 9C2 cells were measured using ELISA. (H) VEGF mRNA expression levels in mock, 9A3, 9C1 and 9C2 cells were determined by real-time PCR analysis. (I, J) Redox status of VEGF in mock and OE cells was examined by Western blotting under reducing (R) or non-reducing (NR) conditions. Reduced form (R) and oxidised form (O) of VEGF are indicated. The ratio of the oxidised form (O) and reduced form (R) obtained in mock cells was set as 1, and the differences in the ratio of the oxidised form (O) and reduced form (R) induced by 9C2 cells were plotted. (K) Western blot analysis of mock, 9A3, 9C1 and 9C2 cells. (L, M) The amount of ROS in 9C2 (bold line) cells was compared with that in mock (thin line) cells. Dotted line and dashed line show unstained cells. The mean fluorescence intensity (MFI) value obtained in mock cells was set as 1, and the differences in MFI induced by 9C2 cells were plotted. *P<0.05, **P<0.01, ***P<0.001, unpaired Student's t-test (vs control).