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. 2020 Jun 30;11:1370. doi: 10.3389/fimmu.2020.01370

Figure 4.

Figure 4

NF-κB Reporter Assays with TLR4, MD-2, and CD14. Human embryonic kidney 293 (HEK293) cells were transfected with plasmid expression vectors for Firefly NF-κB and Renilla luciferase reporters, plus CD14, MD-2, and TLR4 (gray bar); MD-2 and TLR4 (no CD14, orange bar); CD14 and TLR4 (no MD-2, blue bar); or CD14 and MD-2 (no TLR4, green bar). Twenty-four hours after transfection, cells were incubated with media (Control), 10 μM heme, 10 ng/ml LPS, or heme + LPS for 6 h in media containing 1% serum. After 6 h, cells were lysed and luciferase activity was measured and expressed as the ratio of Firefly luciferase NF-κB reporter to Renilla luciferase control. Results are representative of 3 independent experiments run in triplicate. Bars are means + SD. #P < 0.01 control vs. heme, LPS, and heme + LPS. *P < 0.01 TLR4 + MD-2 + CD14 (gray bar) vs. no CD14 (purple bar), no MD-2 (orange bar), and no TLR4 (green bar) for each stimulant, using One Way ANOVA with Holm-Sidac correction.