Fig. 6

SMYD5-mediated inflammatory response depends on upregulation of HK2. A IP-MS was performed to identify SMYD5-interacting proteins. B HEK293T cells were transfected with His-SMYD5 and Flag-HK2 plasmids, followed by co-IP using anti-His antibody. C Immunoblot analysis of HK2 in synovial tissues from patients with RA or OA; HK2 levels were quantified relative to β-actin, n = 3. D FLS were incubated with IL-1β. Immunoblot analysis and quantification of HK2 are shown with β-actin as a loading control, n = 3. E FLS transfected with control or SMYD5 siRNA were incubated with or without IL-1β. Immunoblot analysis and quantification of HK2 are shown with β-actin as a loading control, n = 3. F Lactate levels in the supernatant were measured using a lactate detection kit, n = 3. G–I FLS infected with LV-SMYD5 were challenged with HK2 siRNA (G) or 10 mM 2-DG (H). Immunoblot of SMYD5, HK2, iNOS, and COX2 was quantified relative to β-actin (G, n = 4 or n = 8; H, n = 6). I Lactate levels in the supernatant were measured, n = 3. J FLS pretransfected with control or HK2 siRNA were treated with IL-1β. Immunoblot analysis detected expression and phosphorylation of NF-κB subunits. Phosphorylation of p65, p-IKKα/β, and p-IκBα was quantified, n = 4. K In vitro methyltransferase assay: recombinant HK2 was incubated with SMYD5 and analyzed by SDS-PAGE. L HEK293T cells transfected with Flag-HK2 and His-SMYD5 were immunoprecipitated with anti-Flag and probed for HK2 methylation. M HEK293T cells were cotransfected, and co-IP showed decreased HK2 polyubiquitylation in the presence of SMYD5. Data presented as mean ± SEM, p values calculated by two-tailed Student’s t-test (C) or one-way ANOVA test (D–I), *p < 0.05, **p < 0.01, ***p < 0.001, n.s. means no significance