Expression and localization of S100A14 and S100A16 in human breast cancer cell lines.A, Relative mRNA expression levels measured by real-time RT-PCR. Expression levels were normalized to β-actin levels within the same sample. B, Protein expression of S100A14 and S100A16. Expression levels and subcellular localization were visualized using immunofluorescence staining. Scale bar; 50 μm. C, Subcellular localization of the S100A14 protein on the cell membrane. Z-axis images of confluent MCF7 cells were constructed using confocal laser scanning microscopy. Scale bar; 10 μm. D, Effect of omission of cell permeabilization on the immunofluorescent staining of S100A14. Following fixation with 4% paraformaldehyde, the cells were treated with or without 0.1% Triton X-100 in PBS prior to staining. Scale bar; 50 μm. E, Ca2-independent localization of S100A14 on the cell membrane. MCF7 cells transfected with the S100A14-GFP expression vector were observed by fluorescence microscopy over 180 min after addition of 10 mM EGTA in PBS. Scale bar; 50 μm.