Fig 1: Functional consequences of the inactivation of the ST6Gal-I. a) Immunoblotting of the ST6Gal and its ST6Gal-ICysDM variant in COS-7 cells. Cell transfections were performed by electroporation before SDS-PAGE and immunoblotting with ST6Gal-specific antibodies (anti-CD75) as above. b) Representative figures on SNA lectin binding to the cells transfected separately with the same plasmids. Transfections and lectin staining was carried out as describe above. c) Quantification of the SNA lectin binding to cells using the high content imaging system. The data are shown as relative changes (mean % + SD, n = 3, 70–90000 cells/assay). d) Live cell proliferation rate analysis of COS-7 cells transfected with an empty vector (EV) or with vectors expressing the wild-type ST6Gal-I (WT) or the cysteine double mutant of ST6Gal-I (CysDM). Data are expressed as mean confluence % (±SD (n = 5) using a build-in Incyte™ protocol. The star (*) mark the statistically significant difference (p < 0.05) between relative confluence values of the wild-type ST6Gal-I (WT) and the cysteine double mutant (ST6Gal-ICysDM) at each time point. e) Live cell wound closure analysis of wild type (WT) and double cysteine (CysDM) ST6Gal-I variant expressing cells. The graph shows thee mean confluence (% ±SD (n = 4) at the indicated times after making the wound. Significant changes between confluence values between the wild-type ST6Gal-I (WT) and cysteine double mutant of ST6Gal-I (CysDM) at each time point are marked by stars (p < 0.05*). f) Representative images of the wound closure in COS-7 cells transfected with an empty vector (EV), ST6Gal-I (WT) or the cysteine double mutant of ST6Gal-I (CysDM) at 0 h, 60 h and 120 h time point after introduction of the wound. Scale bar 300 µm. Green color represents the original wound, and yellow color depicts the area of repopulated cells. g) Sensitivity cells to staurosporine-induced apoptosis. Cells transfected with the indicated ST6Gal-I variants were subjected to 5 µM staurosporine for 0–6 h. After fixing, cells were stained with the Hoechst dye before imaging high content imaging system (Operetta, PerkinElmer). Fragmentation index of nuclei was then calculated after image segmentation using the “RMS Nuclear Fragmentation” built-in protocol, in which the coefficient of variance percentage (CV%) of the nuclear intensity is used to calculate the fragmentation index. Healthy nuclei have low CV values whereas fragmented nuclei have higher CV values. The values represent the mean (±SD, n = 3, 15,000 cells each. Statistically significant changes are marked with stars (p < 0.05*, p < 0.01**, p < 0.001***). h-k) The effect of the wild type ST6Gal-I and its double cysteine variant on nuclear and cell morphology. Cells transfected with the indicated constructs were plated on a 96 well plate, fixed immuno-stained with an a-tubulin antibody, and counterstained with the Hoechst dye. After imaging (Operetta, PerkinElmer) and segmentation, in-built “Harmony” protocols were used to calculate nuclear and cytoplasmic areas (h and j) and their roundness (i and k). The data are expressed as the mean % (±SD, n = 3, 15,000 cells each) of the control values (EV = empty vector). No statistically significant changes were detected between the different ST6Gal-I transfectants (p > 0.05).
Fig 2: The effect of hypoxia on the formation of surface exposed disulfide bonds in ST6Gal-I catalytic domain. a) The crystal structure of the human ST6Gal-I catalytic domain (PDB code 4JS2) showing the 3 disulfide bonds between the depicted cysteine residues. Red text refers to surface exposed disulfide bonds while that grey text depicts an internal disulfide bond. b) Bodipy FL-maleimide staining of both ST6Gal-I and B4GalT-I proteins after growing cells for 20 h in normoxic or hypoxic conditions. The figure shows a representative BN-PAGE gel result after labeling of the cells and immunoprecipitation ST6Gal-I and B4GalT-I enzyme proteins with anti-HA tag antibodies from cell lysates prepared from normoxic (N) or hypoxic (H) cells). Band intensities were quantified from digitalized gel images using the ImageJ software. The bars (right) represent fold changes from the normoxic values (set to 1). Statistically significant changes relative to normoxic controls are marked with stars (p < 0.05*, p < 0.01**, p < 0.001***). c) Subcellular distribution of the various ST6Gal-I cysteine mutants. Cells were transfected with the indicated mVenus-tagged enzyme constructs, fixed and co-stained with the anti-GM130 antibody (Golgi marker) before imaging with the fluorescence microscopy. All constructs except the C184A colocalized well with the Golgi marker GM130. d-e) The effect of cysteine mutations on the assembly of the homomeric (d) and heteromeric (e) ST6Gal-I and B4GalT-I complexes. Cell treatments and quantification of the FRET efficiencies were done as above (for details, see the “Material and Methods” section). The bars denote to mean FRET efficiencies (% ± SD, n = 3, 15,000 cells each) from the wild type control values (set to 100%). Only statistically significant changes are shown and marked with stars (p < 0.05*, p < 0.01**, p < 0.001***). f) Quantification of the a-2,6-linked sialic acid levels by SNA lectin staining in control cells and cells expressing the ST6Gal-IWT or the ST6Gal-ICysDM mutant (C353, C406). In brief, 24 h post-transfection cells stained with the Alexa 488-conjugated SNA lectin and with the Hoechst dye to identify cells before quantification of the bound SNA lectin with the Operetta high content imaging system. The data are expressed as the mean SNA intensity/cell (±SD, n = 3, 15,000 cells each) after normalizing the intensities against the ST6Gal-I protein present in the cell lysates (EV Fig. 6). Statistically significant changes relative to control cells (mock-transfected cells) are marked with stars (p < 0.05*, p < 0.01**, p < 0.001***). g) Enzymatic activity of the ST6Gal mutants. Cells transfected with the indicated ST6Gal-I variants were lysed in RIPA buffer 24 h post-transfection, after which their activities were determined as described in the “Materials and Methods” section using asialofetuin as an acceptor. The values shown (bars) are expressed as the mean dpm/10 ng ST6Gal-I protein (n = 3) after normalizing them against the ST6Gal-I protein present (determined by immunoblotting as above). Statistically significant changes relative to control cells (mock-transfected cells) are marked with stars (p < 0.05*, p < 0.01**, p < 0.001***).
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