HTMC cells were treated with PBS or DEX (1×10−4, 1×10−5, 1×10−6 and 1×10−7 M) at 37°C for 48 h. Cell proliferation was analyzed using a Click-iT EdU cell proliferation assay kit according to the manufacturer's instructions. In brief, 3×105 HTM cells were plated on coverslips and incubated with 10 μM EdU solution at 37°C for 16 h. The cells were then fixed with 4% buffered PFA and incubated with 0.5 ml Click-iT reaction mixture for 30 min at room temperature. The coverslides were examined using a Nikon Eclipse 80i microscope system (Nikon, Tokyo, Japan). DAPI was used as a nuclear counterstain (blue) at room temperature for 5 min. EdU-positive cells (green) in 15–20 randomly selected fields were manually counted and the proportion of all cells that were EdU-positive was determined.
For the immunofluorescence analysis of cell morphology, 3×105 HTM cells plated on coverslips were fixed with 4% PFA and permeabilized with Triton X-100. Following the removal of the remaining Triton X-100, the cells were incubated with 5 μg/ml FITC-labeled phalloidin for 1 h. Nuclear staining of the cells was conducted by staining with DAPI at room temperature for 5 min. The coverslides were examined using the Nikon Eclipse 80i microscope system.
For the immunofluorescence analysis of cell morphology, 3×105 HTM cells plated on coverslips were fixed with 4% PFA and permeabilized with Triton X-100. Following the removal of the remaining Triton X-100, the cells were incubated with 5 μg/ml FITC-labeled phalloidin for 1 h. Nuclear staining of the cells was conducted by staining with DAPI at room temperature for 5 min. The coverslides were examined using the Nikon Eclipse 80i microscope system.