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. 2020 May 1;11:2148. doi: 10.1038/s41467-020-15978-3

Fig. 3. High glucose activates MD2-TLR4 signaling in cardiac cells.

Fig. 3

a, b Representative immunoblots showing co-immunoprecipitation of MD2 and TLR4 in H9C2 cells exposed to HG for varying time points (a) and concentration (b) [n = 3]. c H9C2 cells were pretreated with 10 μM MD2 inhibitor L6H21 for 30 min before exposure to HG (33 mM glucose) for 5 min. Representative immunoblots showing co-immunoprecipitation of MD2 and TLR4 [n = 3]. d Western blot analysis showing levels of MD2 protein following transfection of H9C2 cells with MD2 siRNA [si-MD2 = MD2 targeting siRNA, NC = negative control; n = 3]. e Co-immunoprecipitation of TLR4 and MyD88 in H9C2 cells transfected with MD2 siRNA (si-MD2) and exposed to HG (33 mM glucose, 15 min) [n = 3]. f, g Representative blots of IκB and phosphorylation of ERK, JNK, and P38 in H9C2 cells transfected with MD2 siRNA and exposed to HG (33 mM glucose, 15 min). GAPDH and total MAPK proteins served as controls [n = 3]. h, i Tnfa and Il6 mRNA levels in H9C2 cells transfected with MD2 siRNA (si-MD2) and challenged with HG (33 mM glucose, 6 h) [means ± SEM; n = 3 independent examinations]. j Co-immunoprecipitation of TLR4, MyD88, and MD2 in mouse peritoneal macrophages (MPMs). MPMs were pretreated with 10 μM MD2 inhibitor L6H21 for 30 min before exposure to HG (33 mM glucose). MD2-TLR4 complex was assessed following 5 min of HG exposure and TLR4-MyD88 complex at 15 min of HG exposure [n = 3 examinations]. k, l Levels of TNF-α and IL-6 in culture media of MPMs isolated from non-diabetic WT and MD2KO mice. Cells were pretreated with 10 μM L6H21 for 1 h and then exposed to HG (33 mM glucose) for 24 h. TNF-α and IL-6 levels were determined by ELISA [means ± SEM; n = 3 examinations]. Source data are provided as a Source Data file. P-values by one-way ANOVA in h, i, k, and l followed by Tukey’s post hoc test are indicated.