Fig 2: USP10 directly interacts with PLK1.A Flow chart of the USP10 substrate screening process. B A Venn diagram showing that PLK1 is the only substrate of USP10. C The interaction between USP10 and PLK1 was confirmed through molecular docking analysis. D Exogenous CO-IP assay of USP10 and PLK1 in HEK293 cells. E, F Endogenous interaction of USP10 and PLK1 detected via Co-IP assay in PDAC cells. G The direct interaction between USP10 and PLK1 was validated using a GST pull-down assay. H HEK293 cells were transfected with the respective plasmids, and full length and fragments of USP10 were used to pull down full-length PLK1. I HEK293 cells were transfected with the respective plasmids, and full length and fragments of PLK1 were used to pull down full-length USP10.

Fig 3: USP10 maintains PLK1 protein stability by triggering deubiquitination of PLK1.A The change of USP10 and PLK1 protein after transfection with USP10 siRNAs in PDAC cells. B The protein level of USP10 and PLK1 were detected via western blot after transfection with HA-USP10 plasmids. C PANC-1 cells were transfected with si-NC, siUSP10-1, and siUSP10-2 and treated with 20 μM MG132 for 24 h, and the protein level of USP10 and PLK1 were detected through western blot. D PANC-1 cells were transfected with different siRNAs and treated with 10 μg/ml CHX for 0 h, 4 h, 8 h, and 12 h. The change of USP10 and PLK1 proteins were detected. The PANC1 E and MIAPaCa-2 F cells were treated with protein synthesis inhibitor CHX and autophagy inhibitor CQ, and the protein level of PLK1 was detected. G Myc-USP10 and other plasmids were transfected into HEK293 cells and treated with 20 μM MG132 for 6 h. The ubiquitination level of PLK1 was detected via IP assay. H Myc-USP10 (C424A) and other plasmids were transfected into HEK293 cells and treated with 20 μM MG132 for 6 h. The ubiquitination level of PLK1 was detected via IP assay. I The ubiquitinated Flag-PLK1 was purified from HEK293, and GST-USP10 was purified from E. coli BL21 (DE3). Next, the two proteins were incubated in the deubiquitination buffer at 37 °C for 2 h. The ubiquitination level of Flag-PLK1 was detected through western blot.

Fig 4: USP10 affects the autophagy of PDAC cells partially through PLK1.A, B The si-NC, siUSP10-1, siUSP10-2, and the overexpression plasmid of PLK1 were transfected into PDAC cells as required. The EBSS medium was used to activate autophagy. The autophagy-related proteins were detected. C, D PDAC cells were infected with the lentivirus Mcherry-EGFP-LC3B. Next, si-USP10 and the overexpression plasmid of PLK1 were transfected into PDAC cells as required, and the cells were treated with EBSS medium for 8 h before being observed under a confocal microscope. E si-USP10 and the overexpression plasmid of PLK1 were transfected into PDAC cells according to the requirement, and the cells were treated with EBSS medium for 8 h before being observed under a transmission electron microscope. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.