Fig. 4.

LIPUS directly inhibits the TGF-β1-induced fibrotic response and proliferation of FLS. FLS were stimulated with TGF-β1 for 24 ​h and treated with or without LIPUS for 20 ​min (A–D) The protein levels of α-SMA, CTGF and Col Ⅰ were measured by western blot (n ​= ​3 per group) (E) Immunofluorescence assay was taken to measure the expression of α-SMA in FLS. Scale bar:50 ​μm (F) Quantification of α-SMA fluorescence intensity (n ​= ​5 per group) (G) The EdU assay was taken to detect the proliferation of FLS. Nuclei were stained with DAPI (blue) and EdU-positive cells appear red. Scale bar: 100 ​μm (H) Quantification of EdU-positive cells in three randomly selected microscope fields. EdU Positive rate was calculated as total number cells divided by positive cells (n ​= ​3 per group) (I) CCK8 assay was performed to examine the FLS viability. Statistical analysis was performed by one-way ANOVA, and multiple comparisons were performed using Tukey's test. Data are expressed as mean ​± ​SD. ns, not significant, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001, and ∗∗∗∗p ​< ​0.0001.