Effects of IFN-γ and STAT1 on HER2 downregulation mediated by PBMCs in the presence of trastuzumab. (A) IFN-γ detection in the CM by ELISA. BT474 cells were cultured for 48 h in conditions shown at the X-axis and the CM from co-culture with PBMCs in the presence of trastuzumab showed significantly higher level than the other three conditions, **P <0.01. (B) IFN-γ mRNA levels in PBMCs detected by qPCR. PBMCs were incubated in media (control), with trastuzumab (tras), with formaldehyde-inactivated BT474 cells (BT474), and inactivated BT474 cells plus trastuzumab (BT474 + tras) for 24 h before isolation of total RNA for qPCR. **P <0.01. (C) WB detection of HER2 in BT474 cells with and without the addition of an IFN-γ-neutralizing antibody (10 μg/ml) for 48 h as indicated on the top of each lane. (D) Effects of IFN-γ (100 ng/ml) treatment on HER2 expression in BT474 cells. WB detection is shown on the left and mRNA level by qPCR is shown in the bar graph on the right. *P <0.05. (E) WB detection of total pSTAT1 and STAT1 in BT474 cells after culturing with CM collected from four culture conditions as shown on the top of each lane: cancer cells only, cancer cells treated with trastuzumab, cancer cells plus PBMCs, and cancer cells plus PBMCs and trastuzumab. The β-actin band serves as a loading control. (F) Effects of fludarabine on pSTAT1, total STAT1, and HER2 levels as detected by WB. BT474 cells were cultured in 50% CM collected from BT474 cells co-cultured with PBMCs ± trastuzumab, in the presence or absence of fludarabine (100 μM) as indicated on the top. CM, conditioned media; ELISA, enzyme-linked immunosorbent assay; HER2, human epidermal growth factor receptor 2; IFN-γ, interferon gamma; PBMC, peripheral blood mononuclear cell; (p)STAT1, (phosphorylated) signal transducer and activator of transcription 1; WB, Western blotting.