Figure 6. ERK2-mediated PFAS phosphorylation stimulates RAS-dependent tumor growth.

(A) Cell proliferation was measured in HEK293E and A549 ΔPFAS cells stably reconstituted with PFAS-WT or PFAS-T619A (see also Figures S6A and S6B). (B) Soft agar colony formation assay with A549 and A375 ΔPFAS cells stably reconstituted with PFAS-WT or PFAS-T619A. Cell images are at 3x magnification. The relative colony counts are presented as the means ± SDs of biological triplicates. (C) A549 ΔPFAS cells (5 × 10⁶) stably-reconstituted with either WT or T619A-PFAS were injected subcutaneously into athymic nude mice (n = 5 per group). After tumor onset (100 mm³), tumor growth was monitored over time. (D) Normalized peak areas of ¹⁵N-¹³C-labeled purine intermediates measured in HEK293E ΔPFAS cells reconstituted with PFAS-WT or PFAS-T619D cultured in the absence of serum for 15 hours and isotopically labeled with ¹⁵N-¹³C₂-glycine for 1 hour (see also Figures S6D, S6E and S6F). (E) Model of purine synthesis stimulation by the RAS-ERK signaling pathway. * P<0.05 by two-tailed Student’s t test for pairwise comparisons (A-D). The data are representative of at least two independent experiments.