Figure 4.

Inhibition of USP1 by ML323 suppresses proliferation, migration and invasion of OS cell lines. A. The expression of TAZ and downstream genes in Hippo signaling pathway was reduced with the stimulation of ML323 for 48h, as detected by Western Blot. B. The ability of OS cells in forming colonies was weakened after exposing to different dosage of ML323, as determined by colony formation assay. C. Functions of ML323 in osteosarcoma cells proliferation as evaluated by EdU assay. DAPI was utilized to stain the nuclei. Cells in S phase can be stained by both EdU and DAPI. D. 143B and HOS cells were subjected to different dosage of ML323, and then utilized Soft agar colony formation assay to assessed their ability of proliferation. Images were acquired at 1, 5 and 10 days, respectively. E. The expression levels of EMT phenotype-related genes were measured by Western Blot assay after the stimulation with different dosage of ML323 range from 0 to 32 µmol/L as indicated for 48h. F, G. The migration and invasion capacities of 143B cells and HOS cells with the treatment of ML323 were evaluated by transwell migration and invasion assays. H. The wound-healing assay presented that ML323 suppressed migration capacity of 143B and HOS cells in a concentration relative manner. Images were acquired at 0 and 24h respectively for each group. Data represents the means ± SD. The images and data presented were acquired from and represented three independent experiments. P*< 0.05, P** < 0.01, P*** < 0.001 in comparison with the control group.