Fig. 5: ASS1 inhibits TNBC growth by regulating PHGDH stability.

A MDA-MB-468 cells with ASS1 knockout (KO#1 and KO#2) via CRISPR/Cas9 were subjected to MTT assays. Four independent experiments were performed and data are shown as the means ± SD with p-value based on unpaired T-test (n = 4, **p < 0.01). B, C MDA-MB-468 cells with ASS1 knockout (KO#1 and KO#2) via CRISPR/Cas9 were subjected to colony formation assays. Representative images and quantitative results of colonies are shown. Three independent experiments were performed and data are shown as the means ± SD with p-value based on unpaired T-test (n = 3, **p < 0.01). D MDA-MB-468 cells stably expressing the control empty plasmid or Flag-ASS1 were subjected to MTT assays. Four independent experiments were performed and data are shown as the means ± SD with p-value based on unpaired T-test (n = 4, **p < 0.01). E, F MDA-MB-468 cells stably expressing the control empty plasmid or Flag-ASS1 were subjected to colony formation assays. Representative images and quantitative results of colonies are shown. Three independent experiments were performed and data are shown as the means ± SD with p-value based on unpaired T-test (n = 3, **p < 0.01). G Hs578T cells with ASS1 knockdown by shRNA (KD#1 and KD#2) were subjected to MTT assays. Four independent experiments were performed and data are shown as the means ± SD with p-value based on unpaired T-test (n = 4, ***p < 0.001). H Hs578T cells stably expressing the control empty plasmid or Flag-ASS1 were subjected to MTT assays. Four independent experiments were performed and data are shown as the means ± SD with p-value based on unpaired T-test (n = 4, ***p < 0.001). I MDA-MB-468 cells with PHGDH knockout (KO#1 and KO#2) via CRISPR/Cas9 were subjected to MTT assays. Four independent experiments were performed and data are shown as the means ± SD with p-value based on unpaired T-test (n = 4, ***p < 0.001). J, K PHGDH knockout (KO#1 and KO#2) via CRISPR/Cas9 in MDA-MB-468 cells was subjected to colony formation assays. Representative images and quantitative results of colonies are shown. Three independent experiments were performed and data are shown as the means ± SD with p-value based on unpaired T-test (n = 3, **p < 0.01). L MDA-MB-468 PHGDH knockout cells were subjected to MTT assay after ASS1 knockout. Four independent experiments were performed and data are shown as the means ± SD with p-value based on a two-way ANOVA (n = 4, **p < 0.01; n.s., not significant). M, N MDA-MB-468 PHGDH knockout cells were subjected to a colony formation assay after ASS1 knockout. Representative images and quantitative results of colonies are shown. Three independent experiments were performed and data are shown as the means ± SD with p-value based on a two-way ANOVA (n = 3, **p < 0.01; n.s., not significant). O, P MDA-MB-468 cells stably expressing the control empty plasmid or the ASS1 KO, PHGDH KO, ASS1 and PHGDH double knockout plasmids were inoculated subcutaneously into 4 week-old BALB/c nu/nu female mice (n = 6). After 42 days of injection, the mice were sacrificed, and the xenograft tumors were removed. Representative tumor volume (O) and tumor weight (P) are shown. Six independent experiments were performed and data are means ± SD. Significant differences are based on a two-way ANOVA test. (n = 6, *p < 0.05, **p < 0.01, n.s. not significant). Q Immunoblotting analysis of ASS1 and PHGDH protein expression levels in xenograft tumors. The blots shown are from one representative experiment of three replicates.