Figure 2: PA induces MD2-dependent inflammatory injury in cardiomyocytes.

(a,b) H9C2 cells were pretreated with 10 μM L6H9 before stimulation with 500 μM PA for 12 h. Representative images of cell morphology captured by phase microscopy (top panel) and F-actin distribution stained by rhodamine–phalloidin (bottom panel) are shown; n=3; scale bars=20 μm. Quantification of cell size is shown in b. (c) Effects of L6H9 pretreatment on apoptosis induced by 500 μM PA for 24 h in H9C2 cells. Quantification of flow cytometric data for apoptotic cells, determined by flow cytometric analysis of FITC-Annexin V and propidium iodide (PI); n=3. (d) Cytosolic and nuclear protein levels of IκB-α and NF-κB p65 subunit in cells pretreated with L6H9 before stimulation with 500 μM PA for 1 h (representative of n=3). Densitometric quantification of NF-κB pathway activation in cells treated with PA is shown below. (e) Cytokine expression in H9C2 cells following treatment with 500 μM PA for 6 h. Bar graphs showing relative mRNA values for TNF-α, IL-1β, ICAM-1 and VCAM-1 normalized with GAPDH (n=4). (f) Representative western blot with densitometric quantification of ICAM-1 and VCAM-1 in cells treated as in e (representative of n=3). (g) Knockdown of Md2 in H9C2 cells by siRNA transfection. Figure showing protein levels of MD2 in untransfected cells (Ctrl) and in cells transfected with either control/scrambled siRNA (siCtrl) or siRNA targeting Md2 (siMd2; n=4 experiments). (h) Effect of Md2 knockdown on pro-inflammatory gene expression in H9C2 cells stimulated with 500 μM PA for 6 h. Bar graphs showing relative mRNA for TNF-α, IL-6 and IL-1β (GAPDH used as housekeeping gene; n=4 experiments). Data in b–h are reported as mean±s.e.m. and analysed by one-way analysis of varinace, ^#P<0.05, ^##P<0.01, compared with dimethylsulphoxide, Ctrl or siCtrl group; *P<0.05, **P<0.01, compared with PA or siCtrl-PA group.