Figure 1

HER2 downregulation in cancer cells by PBMCs in the presence of trastuzumab. (A) WB detection of HER2 levels in four high HER2 cancer cells BT474, MCF7/HER2, SKBr3, and SKOV-3 cultured in different conditions for 48 h. PBMCs were added at E:T ratio = 10:1 in the presence of trastuzumab (tras) or isotype control IgG (5 μg/ml). Cell lysates were loaded 20 μg per well and β-actin was detected as a loading control. Four treatment conditions are shown for each of the four cancer cell lines and the condition with PBMC and trastuzumab is labeled with the red +. (B) HER2 levels on the cell surface were detected by flow cytometry. Histograms shown in the upper panel are for cancer cells without treatment and the lower panel is for cancer cells with PBMC and trastuzumab treatment. The filled histogram is for cells stained with an isotype antibody control and the open histogram is for cells stained with an anti-HER2 antibody. The mean fluorescence intensity (MFI) of HER2 staining is shown in each histogram. (C) qPCR detection of HER2 mRNA levels in BT474 cancer cells at 8 h, 24 h, and 48 h posttreatment. *P <0.05; **P <0.01. (D) WB detection of HER2 in BT474 cells from co-culture with PBMCs in the presence or absence of trastuzumab under three conditions: no inhibitor control (left); addition of the proteasome inhibitor MG-132 (middle), and addition of the lysosome inhibitor chloroquine (right). HER2, human epidermal growth factor receptor 2; IgG, immunoglobulin G; PBMC, peripheral blood mononuclear cell; WB, Western blotting.