Fig. 4: Impact of SLC33A1 on the formation of 7,9-di-O-acetylated sialosides.
From: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O-acetylation in CASD1 catalysis
a Fluorescence microscopy images of Triton-permeabilized CHO cells stained with the virolectins BCoV-HE0-Fc (7,9-O-Ac-Sia; red) and ICV-HE0-Fc (9-O-Ac-Sia; red). Nuclei were counterstained with DAPI (blue). Representative images from one of three independent experiments are shown. Scale bars: 10 µm. Fluorescence microscopy images of non-permeabilized CHO-WT (b), CHO-ΔSlc33a1 (e) and CHO-ΔCasd1 cells (g) stained with the indicated virolectins. Prior to staining, cells were transfected with empty vector (Mock) or a plasmid encoding human V5-tagged ST8SIA6, alone or in combination with a plasmid encoding human Flag-tagged SLC33A1 or Myc-tagged CASD1. Nuclei were counterstained with DAPI (blue). Scale bars: 10 µm. The expression of ST8SIA6, SLC33A1 and CASD1 was validated by immunofluorescence microscopy as shown in Supplementary Fig. 12. c Virolectin-based flow cytometric analysis of CHO-WT cells before (Mock; black) and after expression of V5-tagged ST8SIA6 (green). Removal of O-acetyl groups by the bacterial sialic acid specific 7,9-O-acetylesterase NanS (dotted lines) was used to validate specific binding of the virolectins BCoV-HE0-Fc and ICV-HE0-Fc. Mock-transfected cells incubated with secondary antibody only were used as controls (gray fill). Virolectin-based flow cytometric analysis of CHO-WT (d), CHO-ΔSlc33a1 (f), and CHO-ΔCasd1 cells (h) transfected with empty vector (Mock) or with a plasmid encoding V5-tagged ST8SIA6 (dark green), alone or in combination with a plasmid encoding SLC33A1 or CASD1 (light green). Results obtained with BCoV-HE0-Fc and ICV-HE0-Fc are shown as open and striped bars, respectively. Data are presented as mean ± s.d. of n = 4 independent experiments (two-way ANOVA followed by Bonferroni’s post-hoc test; ***p < 0.001 and ****p < 0.0001 indicate significant differences between BCoV- and ICV-HE0-Fc-stained cells). Dot plots illustrating the gating strategy are shown in Supplementary Fig. 10. See Supplementary Fig. 11 for the western blot analysis confirming that ST8SIA6 expression is not affected by Slc33a1-deficiency. BCoV bovine corona virus, Fc constant region of human IgG1, HE0 hemagglutinin-esterase carrying an alanine exchange of the active site serine of the esterase domain, ICV influenza C virus, PE phycoerythrin. Source data and exact p values are provided as a Source data file.
