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. 2022 Mar 21;298(5):101853. doi: 10.1016/j.jbc.2022.101853

Figure 3.

Figure 3

Characterizing stably rescued PAICS-knockout HeLa (crPAICS) cells expressing StrepTag-labeled PAICS.A, frontal and oblique view of the crystal structure of human PAICS octamer (2H31). One PAICS monomer is rendered as a ribbon model, with locations of its N and C termini highlighted. B, Western blot analysis of reintegrated PAICS expression in rescued crPAICS cells. The expression of reintegrated PAICS in crPAICS::PAICS-2×Strep is ∼6 fold higher than that in crPAICS::2×Strep-PAICS. All cells except crPAICS were cultured in purine-depleted media. C, Western blot analysis comparing the expression of all six DNPB enzymes: PPAT, GART, PFAS, PAICS, ADSL, and ATIC between crPAICS::2×Strep-PAICS and crPAICS::PAICS-2×Strep cell lines. D, histograms of PAICS expression in single cells, measured from immunofluorescence images. E, cultured under purine-depleted conditions, stably rescued crPAICS::2×Strep-PAICS (doubling time 2.1 ± 0.1 days) proliferates at a faster rate than crPAICS::PAICS-2×Strep (doubling time 2.8 ± 0.2 days); p-value from unpaired t test <0.003. Dashed line indicates initial cell seeding density. Solid lines are fits to an exponential growth curve. ADSL, adenylosuccinate lyase; ATIC, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase; DNPB, de novo purine biosynthesis; GART, phosphoribosylglycinamide synthetase/formyltransferase/phosphoribosylaminoimidazole synthetase; PAICS, phosphoribosylaminoimidazole carboxylase/succinocarboxamide synthetase; PFAS, phosphoribosylformylglycinamidine synthase; PPI, protein–protein interaction; PPAT, amidophosphoribosyltransferase.