Figure 2.
Wild-type pulmonary mesenchymal cells differ from CCR5−/− by their response to CCL4. A: Fluorescent microscopy of wild-type and CCR5−/− mesenchymal cells revealing similar staining for α-SMA. B: Measurement of cell death by annexin V staining demonstrating no difference in survival of wild-type and CCR5−/− cells. Samples were tested in serum-free media alone or with CCL4 (50 ng/ml) or CCL5 (5 ng/ml). Cell debris was excluded with 7-AAD (n = 4). C: Semiquantitative RT-PCR demonstrating the presence of α-SMA in wild-type and CCR5−/− mesenchymal cells. The gel shows cDNA from three separate experiments. The number of cycles for each reaction was chosen to assure linear amplification of the transcript. SDHA was chosen as a loading control based on its high degree of correlation with the amount of mRNA in a wide variety of conditions. D: Semiquantitative RT-PCR demonstrating the expression of CCR5 in wild-type pulmonary mesenchymal cells (n = 3). E: Western blotting for phospho-ERK showing a response to CCL4 (50 ng/ml) in wild-type mesenchymal cells. F: Chemotaxis assay establishing migration of wild-type but not CCR5−/− mesenchymal cells to CCL4. Cells were added to a 5.7-μm chemotaxis plate and incubated for 6 to 8 hours with varying concentrations of CCL4. Migrating cells were detected on the reverse side of the filter using fluorescent microscopy after fixation and staining with 4,6-diamidino-2-phenylindole. Thirty or more high-powered fields were counted (n = 3). Original magnification, ×100.