Fig. 4: ASS1 inhibits PHGDH-mediated de novo serine synthesis. | Cell Death & Disease

Fig. 4: ASS1 inhibits PHGDH-mediated de novo serine synthesis.

From: ASS1 inhibits triple-negative breast cancer by regulating PHGDH stability and de novo serine synthesis

Fig. 4

A MDA-MB-468 cells with ASS1 knockout via CRISPR/Cas9 (KO#1 and KO#2) were subjected to LC‒MS/MS to determine the concentrations of serine and glycine. Representative images of the relative metabolic results are shown in A. Six independent experiments were performed and data are the means ± SD with p-values based on unpaired T-test. (n = 6, **p < 0.01). B Hs578T cells with ASS1 knockdown by shRNA (KD#1 and KD#2) were subjected to LC‒MS/MS to determine the concentrations of serine and glycine. Representative images of the relative metabolic results are shown in B. Six independent experiments were performed and data are the means ± SD with p-values based on unpaired T-test. (n = 6, *p < 0.05, **p < 0.01). C, D MDA-MB-468 and Hs578T cells stably expressing the control empty plasmid or Flag-ASS1 were subjected to LC‒MS/MS to determine the concentrations of serine and glycine. Representative images of the relative metabolic results are shown in C and D. Six independent experiments were performed and data are the means ± SD with p-values based on unpaired T-test. (n = 6, *p < 0.05, **p < 0.01). E PHGDH, ASS1, PHGDH and ASS1 were knocked out by CRISPR/Cas9 in MDA-MB-468 cells, and the cells were subjected to LC‒MS/MS to determine the concentrations of serine and glycine. Representative images of the relative metabolic results are shown in (E). Six independent experiments were performed and data are the means ± SD with p-values based on two-way ANOVA. (n = 6, *p < 0.05, **p < 0.01, n.s., not significant). F Hs578T cells with PHGDH, ASS1, PHGDH and ASS1 knocked down by shRNA were subjected to LC‒MS/MS to determine the concentrations of serine and glycine. Representative images of the relative metabolic results are shown in F. Six independent experiments were performed and data are the means ± SD with p-values based on two-way ANOVA. (n = 6, *p < 0.05, **p < 0.01, n.s., not significant). G MDA-MB-468 cells cultured in medium containing 25 mM [U-13C] D-glucose with 0.4 mM serine/glycine were lysed for stable isotope flux analysis. Flag-ASS1 decreased the incorporation of 13 C into serine and glycine in MDA-MB-468 cells, which was largely abolished by PHGDH knockout. Three independent experiments were performed and data are the means ± SD with p-values based on two-way ANOVA (n = 3, **p < 0.01, ***p < 0.001, n.s., not significant). H MDA-MB-468 cells cultured in medium containing 25 mM [U-13C] D-glucose with 0.4 mM serine/glycine were lysed for stable isotope flux analysis. Neither Flag-ASS1, PHGDH knockout, nor Flag-ASS1 and PHGDH knockout significantly affected the incorporation of 13 C into lactic acid in MDA-MB-468 cells. Three independent experiments were performed and data are the means ± SD with p-values based on one-way ANOVA. (n = 3, n.s., not significant).

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