Fig. 4: GALA activates O-glycosylation and cell surface exposure of CNX in arthritis primed SF.

a VVL lectin pulldown and CNX co-precipitation with extract from SW982 ER-2Lec inducible cells, untreated (UT), or subjected to stimulation with arthritis associated cytokines (CYTO) and cartilage ECM. Actin was used as a loading control. b Quantification graph of western blot. Data are shown as mean ± SEM and representative of three independent experiments. **, p < 0.01 (p = 0.002; UT vs CYTO ECM, −dox condition and p = 0.0073; −dox vs +dox, CYTO ECM treatment); ns not significant (p = 0.413; UT vs CYTO ECM, +dox condition and p = 0.9447; −dox vs +dox, UT) (One-way ANOVA test). c Representative flow cytometry histogram plots with SW982 ER-2Lec inducible cells, x-axis depicts CNX cell surface positive signal and y-axis marks cell counts. d Quantification graph of percentage of cells expressing cell surface CNX. Data are shown as mean ± SEM from four replicate wells from two independent experiments. ***, p < 0.001 (p = 0.0002; UT vs CYTO ECM, −dox condition and p = 0.0002; −dox vs +dox, CYTO ECM treatment); ns, not significant (p = 0.366; UT vs CYTO ECM, +dox condition and p = 0.4863; −dox vs +dox, UT) (One-way ANOVA test). e Flow cytometry histogram plots with patient derived SF untreated (UT), or subjected to stimulation with arthritis associated cytokines (CYTO) and cartilage ECM, x-axis depicts CNX cell surface positive signal and y-axis marks cell counts. A control surface stain with isotype antibody is presented. f Quantification graph of percentage of cells expressing cell surface CNX. Data are shown as mean ± SEM of three replicate wells, representative of two independent experiments. *, p < 0.05 (p = 0.0296; UT HSCF vs RASF); ****, p < 0.0001 (CYTO ECM HSCF vs OASF or RASF) (One-way ANOVA test).