Fig 2: Schematic of our study illustrating the role of PUS10 in RCC (By Figdraw)

Fig 3: PUS10 inhibits the migration of RCC in a catalytic-independent manner. A Overexpression of PUS10 was verified by qRT?PCR using GAPDH as a reference. B Western blot and dot blot assay were performed to verify the construction of wildtype and mutant PUS10 overexpressing models in vitro. C Transwell assays were conducted to evaluate the migration inhibition effect of wild-type and catalytically incompetent PUS10. Migrating cells in three replicate experiments were counted and are presented as the mean ± SD in the histogram. D Wound healing assay validates the similar impact of wild-type and mutant PUS10 ectopic expression on the migratory ability of RCC cells. E Transwell assays showed that supplementation with either wild-type or mutant PUS10 could reverse the promotion of RCC migration induced by PUS10 silencing with siRNA. Migrating cells in three replicate experiments were counted and are presented as the mean ± SD in the histogram. *P < 0.05, **P < 0.01, ***P < 0.001; ns not significant

Fig 4: Silencing of PUS10 promotes RCC migration in vitro and in vivo. A qRT?PCR assay revealed that PUS10 was knocked down in 786-O and Caki-1 cells after the transfection of siRNAs. Three independent experiments are shown as the mean ± SD, and GAPDH was used as a reference. B Representative western blot images showing the silencing efficiency of PUS10 in RCC cell lines. C Transwell assays and wound healing assays were performed to measure the migration ability in vitro after PUS10 depletion. Migrating cells in three replicate experiments were counted and are presented as the mean ± SD in the histogram. D Wound healing assay validated the promoted migratory ability of 786-O and Caki-1 cells after PUS10 knockdown. A representative of three replicated experiments is shown. The wound healing rate was calculated and presented as the mean ± S.D. E Luciferase-tagged ACHN cells with or without PUS10 knockdown were constructed and injected into the tail vein of nude mice. Bioluminescent images were taken after 8 weeks with signal intensities (photons/s/cm2/sr) qualified using the IVIS system. *P < 0.05, **P < 0.01, ***P < 0.001; ns not significant

Fig 5: The NUDC/cofilin1-dependent cytoskeleton is involved in the PUS10-induced migration suppression of RCC. A KEGG enrichment analysis of the genes possessing a negative correlation with PUS10 in KIRC. B, C The efficiency of NUDC silencing was demonstrated at the mRNA and protein levels. D Transwell assays revealed that NUDC depletion abrogated the promoted migration caused by PUS10 knockdown in Caki-1 and 786-O cells. Migrating cells in three replicate experiments were counted and are presented as the mean ± SD in the histogram. E Wound healing assays showed that NUDC depletion reversed the enhanced wound healing rate induced by PUS10 knockdown in Caki-1 and 786-O cells. Three replicate experiments were conducted, and the results are presented as the mean ± SD in the histogram. F Representative images of immunostaining using anti-cofilin1 antibody and phalloidin are shown, showing the impact of PUS10 on the cofilin1-dependent cytoskeleton. G Western blot assays showed that the increased expression of Coflin1 induced by PUS10 knockdown was rescued by NUDC depletion in Caki-1 and 786-O cells. *P < 0.05, **P < 0.01, ***P < 0.001; ns not significant