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. 2023 Jun 22;6(9):e202302066. doi: 10.26508/lsa.202302066

Figure 5. CTPS1 is more efficient than CTPS2 in restoring the proliferation of CTPS1-KO and CTPS1+2-KO HEK cells.

Figure 5.

(A, B, C, D) CTPS1-KO or CTPS1+2-KO cells were transfected with linearized C1 EGFP vectors containing GFP–CTPS1 or GFP–CTPS2. Cells were then maintained in culture without cytidine and sorted on GFP expression. (A) Histograms of GFP expression after sorting and culture in the absence of cytidine. (B) Western blots for CTPS1 and CTPS2 expression in cell lysates. Actin expression as a loading control. (C) Confluency curves as percentages (%) showing the proliferation. Confluency measurement using an IncuCyte Zoom system. Cells were seeded for 24 h, then untreated or maintained in the presence or absence of cytidine (200 µM) or 3-deaza-uridine with the indicated concentrations. (D) CTPS activity measured in cell extracts of CTPS1-KO cells reconstituted with GFP alone, GFP–CTPS1, or GFP–CTPS2. Means with SEM. Data from four independent experiments with replicates. Two-tailed unpaired t tests againt HEK values; n.s., no significance; **P < 0.01; ***P > 0.001. (A, B, C) Data of one representative experiment of three independent experiments in (A) and four in (C) except for CTPS1+2-KO cells with GFP–CTPS2 only tested two times.