Figure 1. LPS activates TLR4 signaling in A253 cells.

A253 cells were purchased from American Type Culture Collection. Cells were cultured in McCoy’s 5A (Modified) Media containing 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin in 6 well plates. (A) A253 cells (3.5 × 10⁵) were harvested, and relative TLR4, Ly96, CD14, and MyD88 transcript levels were quantified by real time PCR. Each sample was analyzed in duplicate and normalized expression relative to GAPDH is shown. (B) A253 cells were harvested and cultured in the presence or absence of LPS (10 µg/mL) derived from Salmonella typhimurium for 24 h. Tissue was fluorescently stained with isotype control (grey shading) or TLR4 antibody. The red line represents unstimulated cells and the blue line indicates LPS treated cells. (C) A253 cells (3.0 × 10⁵) were incubated in the presence or absence LPS (10 µg/mL) for 24 h. Cells were lysed and western blots performed for MyD88. All samples were normalized to GAPDH. (D) A253 cells (3.0 × 10⁵) were stimulated with LPS (10 µg/mL) for the indicated times. Phosphorylated p65 was normalized to total p65 levels. Results of at least three independent experiments are shown (min = minutes).