Fig. 2: SPA and LM-2I directly target ASS1.

a Chemical structures of SPA (1) and its reductive form SPAH (3). b Crystal violet assay for the viability of MCF-7 cells treated with different concentrations of SPA and SPAH (mean ± s.d., n = 3 biologically independent experiments). c Chemical structures of biotin-labeled SPA [Biotin-SPA (4)] and biotin-labeled SPAH [Biotin-SPAH (5)]. d Crystal violet assay for the viability of MCF-7 cells treated with different concentrations of biotin-labeled SPA [Biotin-SPA (4)] and biotin-labeled SPAH [Biotin-SPAH (5)] (mean ± s.d., n = 3 biologically independent experiments). MCF-7 cell lysates were incubated with biotin or Biotin-SPA in the presence or absence of LM-2I at 4 °C overnight, followed by pulling-down with streptavidin magnetic beads. The proteins bound to the magnetic beads were separated by SDS-PAGE, followed by silver staining (e) or by western blot using ASS1 antibody (f) (n = 3 biologically independent experiments). g Fluorescent staining of MCF-7 cells incubated with or without Biotin-SPA for 4 h (n = 3 biologically independent experiments). Scale bars are 20 µm. h The recombinant ASS1 protein was incubated with Biotin-SPA for different time periods as indicated, followed by western blot using streptavidin-HRP or ASS1 antibody (n = 3 biologically independent experiments). i The recombinant ASS1 protein was incubated with Biotin-SPA in the absence or presence of LM-2I for 1 h at room temperature, followed by western blot using streptavidin-HRP or ASS1 antibody (n = 3 biologically independent experiments). Source data are provided as a Source Data file.