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. 2024 Aug 12;5(8):e684. doi: 10.1002/mco2.684

FIGURE 5.

FIGURE 5

USP22 activates PI3K/Akt pathway via SIRT1/PTEN axis. (A) Protein–protein interactions (PPI) analysis containing USP22, USP22 binding proteins and proteins involved in modulating PI3K–Akt signal pathways. Proteins interacting with USP22 were identified from immunoprecipitation and mass spectrometry (IP‐MS) analysis. (B) Western blotting analysis showing SIRT1 and EZH2 protein expression in USP22‐inducible cells with doxycycline (0–1 µM, wedge). (C) The peak map of SIRT1 acquired from the mass spectrometry. (D) A375 cells were transfected with vectors encoding Flag‐tagged USP22 and Flag‐tagged SIRT1 respectively. Protein lysates then were harvest for immunoprecipitated with anti‐Flag beads, then immunoblotted with the SIRT1 and USP22. (E) Protein lysates of control and USP22‐overexpressing (USP22) A375 cells were treated with cycloheximide (CHX) (100 µg/mL) for the indicated time points, followed by immunoblotting with SIRT1. (F) Quantification of the data from (E) (n = 3 biologically independent samples). (G) Protein lysates of control and USP22‐knockout (sgUSP22) A375 cells were treated with DMSO, MG132 (10 µM) and CQ (20 µM) for 6 h respectively, and followed by immunoblotting with SIRT1 and USP22. (H) Protein lysates of USP22‐overexpressing (USP22) and USP22‐knockout (sgUSP22) A375 cells harvested after MG132 (10 µM) treatment (6 h) for immunoprecipitated with ubiquitin (Ub) antibody plus protein A/G beads and immunoblotted with SIRT1. (I) Immunoblot analysis of 293T cells transfected with Flag‐SIRT1, HA‐tagged ubiquitin, HA‐tagged K6‐linked ubiquitin, HA‐tagged K11‐linked ubiquitin, HA‐tagged K27‐linked ubiquitin, HA‐tagged K29‐linked ubiquitin, HA‐tagged K33‐linked ubiquitin, HA‐tagged K48‐linked ubiquitin, and HA‐tagged K63‐linked ubiquitin with or without USP22, followed by treatment with MG132 and IP with anti‐Flag beads, then detected with anti‐HA. (J) USP22‐overexpressing (USP22) and control A375 cells were transfected with SIRT1 siRNAs. Protein lysates were harvested for immunoprecipitated with PTEN antibody plus protein A/G beads and immunoblotted with acetyl‐lysine. (K) Western blot analysis of PI3K/Akt/mTOR changes in indicated control and USP22‐overexpressing (USP22) cells after transfection with PTEN siRNAs. (L) Spearman's correlation between vimentin (VIM) and SIRT1 based on data from Xiangya cohort and TCGA‐SKCM.