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. 2022 Jun 2;11(1):31. doi: 10.1038/s41389-022-00404-8

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© The Author(s) 2022, corrected publication 2023

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Fig. 4 — A Representative images of silver-stained protein bands and mass spectrometry (MS) analysis. The result was obtained after three times’ repeat. B Gene signature associated with CYCS in the USP53^low and USP53^high groups of TCGA-LIHC dataset as identified by GSEA. Three hundred seventy-one samples were divided into USP53^low and USP53^high groups according to the median of USP53 expression level. Number of permutations:1000. C Representative exogenous immunofluorescence images showing the co-localization of USP53 and CYCS. Scale bar:10 μm. D Exogenous co-immunoprecipitation showing interaction of USP53 and CYCS. The results were repeated three times. E Endogenous immunofluorescence images showing the co-localization of USP53 and CYCS and their position relationship with mitochondria (stained by MitoTracker) in Huh-7 and HCCLM3 cells. Scale bar:10 μm. F Proximity ligation assay (PLA) showed the direct connection of USP53 and CYCS, which takes place outside of mitochondria. Scale bar: 10 μm. G Mitochondrial and cytoplasmic proteins were isolated and detected separately, revealed the subcellular location of USP53 were mostly in cytoplasmic in Huh-7 cell line. The experiment was repeated three times. H GST pull-down assay showed the direct binding of USP53 and CYCS in HEK-293T cells. Pure GST was used as control. The experiment was repeated three times.