Fig 1: CDC42 activated NF-?B p65 to regulate GLS1 expression and drove GLS1 microvesicle secretion via IQGAP1. (A) WB analysis of GLS1 and CDC42-GTP protein in trastuzumab-resistant cells with different CDC42 mutant. (B) GLS1 enzyme activity of trastuzumab-resistant cells with different CDC42 mutant. (C) WB analysis of Flotillin 2, Alix, TGM2, GLS1 and I?Ba protein in trastuzumab-resistant cells with different CDC42 mutant and microvesicles isolated from those cell's supernatant. (D) GLS1 enzyme activity of microvesicles isolated from trastuzumab-resistant cells with different CDC42 mutant. (E) Top, genes significantly positively correlated with CDC42 and GLS1 were analyzed; Bottom, the differential genes between trastuzumab-sensitive cells and trastuzumab-resistant cells, which positively correlated with CDC42 and GLS1, were enriched into NF-?B pathway (NES = 1.510, FDR = 0.001). (F) WB analysis of p-NF-?B p65 protein in nucleus and cytoplasm of trastuzumab-sensitive and trastuzumab-resistant cells. (G) Trastuzumab-resistant cells were harvested for ChIP assay to detect the enrichment of NF-?B p65 around the GLS1 promoter. Immunoprecipitated DNA was analyzed by qRT-PCR with specific primers. Anti-H3 antibody was used as the positive control. Anti-IgG antibody was used as the negative control (n = 3). (H) WB analysis of GLS1, p-NF-?B p65 and CDC42-GTP protein in trastuzumab-resistant cells treated with or without NF-?B agonist PMA (50 nmol/L, 48h) and CDC42-GTP inhibitor ZCL278 (10 µmol/L, 48h). (I) WB analysis of IQGAP1, GLS1, Flotillin 2, Alix, TGM2 and I?Ba protein in trastuzumab-resistant cells transfected with control plasmid or siRNA of IQGAP1 and microvesicles isolated from those cell's supernatant. (J) Co-IP assay was used to detect the protein–protein binding of IQGAP1 and GLS1. Left, GLS1 antibody coprecipitating IQGAP1. Right, IQGAP1 antibody coprecipitating GLS1. Input, protein expression in cell lysates. IgG, negative control. IP, expression of compound coprecipitated by GLS1 or IQGAP1 antibody. The data are presented as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, nonsignificant.Abbreviations: TR, trastuzumab-resistant; GSEA, gene set enrichment analysis; NES, normalized enrichment score; CDC42, cell division cycle 42; GLS1, glutaminase-1; NF-?B, nuclear factor kappa B; IQGAP1, IQ motif containing GTPase activating protein 1; TGM2, transglutaminase 2; I?Ba, inhibitor kappa B alpha; ChIP, Chromatin Immunoprecipitation; PMA, phorbol 12-myristate 13-acetate; Co-IP, co-immunoprecipitation; SD, standard deviation.