Fig. 2

Upregulation of USP22 by USP7 inhibition through SP1 degradation. FT671 treatment significantly elevated A. USP22-promoter driven firefly luciferase reporter activity and B. Dynamic increase of USP22 mRNA level in both A549 and H1299 lung cancer cells, increase of USP22 mRNA reached maximum at 12h posttreatment, *p<0.05, ** p<0.01, compared to Vehicle. C. FT671 treatment induced a significant decrease of SP1 and β-Catenin in both A549 and H1299 cancer cells. D. The upregulation of USP22 by USP7 knockdown is dependent on decrease in SP1 transcriptional activity. Western blot analysis showed that reintroduction of SP1 in USP7-silenced H1299 cells significantly attenuated the upregulation of USP22 by USP7 knockdown. (Blk: without plasmid transfection, Ctrl: Control plasmid, SP1: RSV-SP1 plasmid). E. FT671 promoted the degradation of SP1 in A549 cells. A549 cancer cells were pretreated with FT671 for 4 hours, and then treated with 50 µg/ml cycloheximide (CHX) for 0-24h, and the protein was used for Western blot analysis