Fig. 6: ASS1 is indeed the target of SPA and LM-2I.

a Crystal violet assay for the viability of MDA-MB-231 cells treated with LM-2I or LM-2I pre-incubated with different concentrations of ASS1 recombinant protein (mean ± s.d., n = 6 biologically independent experiments). ***P < 0.001, NS: not significant (P > 0.05) from two-tailed unpaired Student’s t-tests. b Western blot for ASS1 expression in control or stable ASS1 knockout (ASS1-KO) MDA-MB-231 cells (top). Crystal violet assay for the viability of control and ASS1-KO MDA-MB-231 cells treated with LM-2I (bottom) (mean ± s.d., n = 3 biologically independent experiments). c Western blot for ASS1 expression in control or stable ASS1 knockdown (ASS1-KD) MCF-7 cells (top). Crystal violet assay for the viability of control and ASS1-KD MCF-7 cells treated with LM-2I (bottom) (mean ± s.d., n = 3 biologically independent experiments). d Western blot for ASS1 expression in control or stable ASS1 overexpression (ASS1-OE) MDA-MB-231 cells (top). Crystal violet assay for the viability of control and ASS1-OE MDA-MB-231 cells treated with LM-2I (bottom) (mean ± s.d., n = 3 biologically independent experiments). e The correlation between the relative ASS1 expression in cells and the IC₅₀ of these cells treated with LM-2I (n = 3 independent experiments from n = 14 cell lines). P values of mean ± s.d. were determined by Pearson Coefficient. f LC-MS analysis of ASP, UMP, CMP, and TMP in MDA-MB-231 cells treated with SPA, LM-2I or vehicles for 24 h (mean ± s.d., n = 6 biologically independent experiments). **P < 0.01, ***P < 0.001 from two-tailed unpaired Student’s t-tests. g Crystal violet assay for the viability of MDA-MB-231 cells preincubated with pyrimidines or purines, followed by treatment with SPA or LM-2I (mean ± s.d., n = 6 biologically independent experiments). ** P < 0.01, ***P < 0.001, NS: not significant (P > 0.05) from two-tailed unpaired Student’s t-tests.Source data are provided as a Source Data file.