Fig. 6: Studying the effects of mutations in the CS polymerase complex on cellular CS biosynthesis.

a Schematic illustration of the in-cellulo complementation assay. Knockout (KO) of the CHSY1 and CHSY3 genes via CRISPR/Cas eliminates the production of CS chains. KO cells are then co-transfected with either CHSY1 and CHPF or CHSY3 and CHPF encoding plasmids, and cell surface CS levels quantified. b Complementation of CHSY1/CHSY3 KO cells with CHPF wild-type and CHSY1 wild-type or mutant constructs. Cell surface CS levels in non-transfected cells (blue bars) and co-transfected cells (green bars) were quantified by flow cytometry using a primary anti-CS antibody and an Alexa Fluor 488-labeled secondary antibody. Results were compared to wild-type HEK293-6E cells. Experiments were performed in triplicate (n = 3). Error bars indicate standard deviation from the mean values. A one-sided paired Student’s t-test was used to determine p-values (*<0.05 and **<0.01) or more precisely CHSY1 WT vs D171N/D173N (0.0030), H304A (0.0030), D631N/D633N (0.0026) and H744A (0.0031). c Complementation assay as described in (b), but with CHSY3-CHPF (orange bars). Experiments were performed in triplicate (n = 3). Error bars indicate standard deviation from the mean values. A one-sided paired Student’s t test was used to determine p-values (*<0.05 and **<0.01) or more precisely CHSY3 WT vs D261N/D263N (0.0439), H394A (0.0442), D718N/D720N (0.0438) and H831A (0.0437). Source data is provided. Source data are provided as a Source Data file.