Fig. 4: HOG-LDL causes MIO-M1 cell apoptosis by enhancing the NOX4-TLR4 interaction in a MD2-dependent manner.

a–c MIO-M1 cells were pretreated with or without MD2 inhibitors for 2 h and exposed to HOG-LDL for 15 min. Protein samples were immunoprecipitated with MD2 antibodies (a) or TLR4 antibodies (b, c). Associated proteins were detected using immunoblotting. d MIO-M1 cells were transfected with siRNA against MD2 (siMD2) or negative control (NC) siRNA. The levels of MD2 were measured after 24 h. GAPDH was used as a loading control. e, f Cells transfected with siMD2 or NC were exposed to HOG-LDL for 3 h, and ROS levels were measured by DCFH-DA. e Shows representative flow cytometry data. The quantification of ROS levels is shown in (f). g MIO-M1 cells were transfected with siMD2 or NC and exposed to HOG-LDL for 12 h. The number of apoptotic cells was determined using flow cytometry. h Cells were transfected with siMD2 or NC and exposed to HOG-LDL for 15 min. Protein samples were immunoprecipitated with TLR4 antibody, and NOX4 was detected by immunoblotting. i–l Cells were transfected with siMD2 or NC and exposed to HOG-LDL for 12 h to analyze NOX4 (i), cleaved PARP (j), and GRP78 (k) or 2 h to analyze IκBα (l). GAPDH was used as a loading control. Densitometric quantification is shown in the lower panels. Quantitative data are presented as the mean ± SEM, n = 3; ^#P < 0.05; ^##P < 0.01; ^###P < 0.001 compared to the untreated NC; *P < 0.05; **P < 0.01 compared to the HOG-LDL-treated NC.