Fig. 4: Cells exclusively expressing PRPS1 display metabolic defects and decreased cellular proliferation.
A Western blot validating CRISPR-Cas9-generated isogenic knockout cell lines. B Proliferation of NIH3T3 parental and knockout cell lines generated in (A) (n = 3 technical replicates). C Western blot validating HRASG12V-overexpression in NIH3T3 parental and P2/AP1/AP2 KO cell lines. Phospho-MAPK (Erk1/2) (T202/Y204) used as a marker for activation of signaling pathways upon HRASG12V overexpression. D Representative images from soft agar colony formation assay performed in NIH3T3 parental and P2/AP1/AP2 KO cells expressing HRASG12V. Scale bar, 500 μm. E Quantification of colonies from (D) (n = 3 experimental replicates). F Oxygen consumption rate (OCR) measured by Seahorse ATP Rate Assay in NIH3T3 parental and P2/AP1/AP2 KO cell lines (n = 8 technical replicates). G OCR measured by Seahorse mitochondrial stress tests in NIH3T3 parental and NDI1-expressing P2/AP1/AP2 KO cell lines (n = 7 technical replicates). H Proliferation of nucleoside supplemented and NDI1 expressing NIH3T3 P2/AP1/AP2 cell lines (n = 3 technical replicates). I13C6-glucose metabolic labeling performed in NIH3T3 parental and P2/AP1/AP2 KO cell lines for 30 min and 5 h. Unlabeled, 13C-labeled (30 min), and 13C-labeled (5 h) samples are represented in brown, green, and blue colors, respectively. 13C-enrichments quantified from 1H-NMR spectra (n = 3 experimental replicates). J Western blot analysis of SEC fractions collected from NIH3T3 P2/AP1/AP2 KO native whole-cell lysates. Cell lysates were fractionated on a Superose 6 Increase 3.2/300 column. In the pictogram below SEC immunoblots, the double circle with dotted inner circle denotes multiple copies of PRPS1 forming homo-oligomers. Data are represented as mean ± SD for (B, E–H) and mean ± SEM for (I). Statistical comparisons made using one way ANOVA followed by Tukey’s HSD post hoc test (E, I). Source data are provided as a Source Data file.
