Fig 2: The rule between IARS and PTEN in the presence of isoleucine. a Immunofluorescent staining of 293 T cells transfected with plasmids expressing Flag-PTEN-EGFP or Flag-PTEN-EGFPK13,289E for 4 h, and then treated with 50 × isoleucine for 12 h, scale bar represents 50 µm (n = 3); b Proteins most directly associated with isoleucine in proteins precipitated by anti-Flag-PTEN; c Pearson correlation between IARS1 and PTEN in LUAD from TCGA database; d A549 cells treated with 50 × isoleucine for 48 h were immunoprecipitated with anti-PTEN antibody and subjected to Western blot analysis. IgG was used as negative control (n = 3); e Relative expression level of Iars1 mRNA in LLC tumor tissues (n = 3); f IARS1 protein level in LLC tumor tissues (n = 3); g IARS1 protein level in H1975 cells treated with different concentrations of isoleucine for 48 h (n = 3); h IARS1 immunofluorescent staining in H1975 cells, scale bar represents 50 µm (n = 3); i Immunofluorescent colocalization between IARS1 and PTEN in A549 cells (n = 3); j A549 cells were treated with 50 × isoleucine for 48 h and subjected to Western blot analysis after extraction of cytoplasmic (Cyto) and nuclear (Nucl) proteins (n = 3); k Western blot analysis of cytoplasmic (Cyto), nuclear (Nucl), whole-cell lysates (WCL) in A549 cells transfected with plasmids expressing Flag-IARS1-EGFP for 4 h, and then treated with 50 × isoleucine for 12 h (n = 3); l Growth curve of stably IARS1-knockdown (IARS1) A549 cells and wild type (WT) A549 cells treated with 50 × isoleucine (n = 6); m The schematic diagram of the anticancer mechanism of isoleucine-mediated interaction between IARS and PTEN. ns no significant difference; *** p < 0.001. Different letters, p < 0.05, same letters, no significant difference. Different letters, p < 0.05, same letters, no significant difference

Fig 3: Isoleucine promotes the stability of nuclear PTEN based on the PTEN nuclear accumulation. a LLC cells and H1975 cells were treated with 50 × isoleucine for 48 h and subjected to Western blot analysis after extraction of cytoplasmic (Cyto) and nuclear (Nucl) proteins (n = 3); b PTEN immunofluorescent staining in H1975 cells and LLC cells, scale bar represents 50 µm (n = 3); c A549 cells and H1975 cells were treated with 50× isoleucine for 48 h and subjected to immunoprecipitation with an anti-PTEN antibody, followed by Western blot analysis with ubiquitination antibodies as indicated (n = 3); d Stably overexpressed Flag-PTEN A549 cells were treated with 50 × isoleucine for 48 h and cytoplasmic (Cyto) and nuclear (Nucl) proteins were immunoprecipitated with anti-Flag antibody followed by Western blot analysis using ubiquitination antibody (n = 3); e A549 cells were treated with different concentrations of isoleucine for 48 h, and related proteins involved in PTEN ubiquitination were analyzed by Western blot (n = 3)