Fig 2: Knockdown of uPAR promotes fibrosis progression in vitro. (A, B) The knockdown efficiency of uPAR-siRNA in disease-derived fibroblasts was confirmed by western blot and qPCR. (C, D) mRNA level of α-SMA and type I collagen in three groups of cells. (E–H) Immunofluorescence staining of α-SMA and type I collagen in three groups of cells and their integrated density. scale bars, 100 μm, 20μm. (I) The proliferation ability after knocking down uPAR was evaluated by the cell counting kit 8. (J) Effect of knocking down uPAR on the contractility of fibroblasts. (K) The migration ability of fibroblasts after knocking down uPAR was evaluated by wound-healing assay. **P < 0.01, ***P < 0.001, and ****P < 0.0001 when compared between the denoted groups.

Fig 3: Biological Effects of MIR143 Replacement or UPAR Knockdown In Vitro(A) Inhibition of PC-3 cell proliferation upon MIR143 replacement or UPAR knockdown. PC-3 cells were transfected with pre-MIR143, siRNA against UPAR, or the appropriate negative controls. At the indicated time points, cell proliferation was determined by BrdU incorporation. (B) Effects on cell density upon single or combined transfection of MIR143 and/or si-UPAR in DU-145 cells. Scale bar, 200 μm. Decreased numbers of cells were also associated with increased percentages of dead cells in (C) DU-145 and (D) PC-3 cells, as determined by propidium iodide staining in Celigo Imaging Cytometry. Data are presented as mean ± SEM (A) or as mean ± SD (C and D); *p < 0.05; **p < 0.03.

Fig 4: Ulinastatin reduces the expression of uPAR, NFATc1 and osteoclast marker genes induced by RANKL. C represents control group, R represents RANKL group, R + ulinastatin represents RANKL + 800 units/mL ulinastatin group. (A) Ulinastatin reduces RANKL-induced mRNA expression of cathepsin K, Trap, Rank, NFATc1, and uPAR. BMMs were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL), and treated with or without ulinastatin (800 units/mL) for 4 days. mRNA expression was detected by qRT-PCR. The qRT-PCR experiments have been repeated with different RNA preparations for 3 times independently. Data are represented as mean ± SD. ∗P < 0.05 and ∗∗P < 0.01. (B,C,D) Ulinastatin reduces RANKL-induced protein expression of uPAR, cathepsin K and Trap. BMMs were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL), and treated with or without ulinastatin (800 units/mL) for 2 or 4 days. Protein expression levels of uPAR, cathepsin K and Trap were examined by western blotting at the indicated times. The amount of loaded protein was 25 μg. The experiment was performed three times independently and GAPDH was used as a loading control. ∗P < 0.05, ∗∗P < 0.01 versus RANKL group.

Fig 5: Knockdown of uPAR decreased RANKL-induced osteoclast and actin ring formation. NC represents siRNA negative control, Si-1, Si-2, and Si-3 represent siRNAs that silence three different fragment of uPAR. BMMs were seeded in 96-well plates and transfected with three siRNAs and NC siRNA, respectively, and cultured with M-CSF (30 ng/mL) and RANKL (50 ng/mL) for 5 days. (A) Immunofluorescence and (B) TRAP staining was performed then. The experiment was performed three times independently.