Figure 6.

Alt a 1 activates toll-like receptor (TLR)2/4-dependent NF-κB signaling in engineered human embryonic kidney (HEK) cells. 2.5 × 10⁴ HEK-Blue hTLR4 (A,B), HEK-Blue hTLR2 (C), and HEK-Blue null cells (A–C) were plated in 96-well plates in 200 µL DMEM + 1% Pen/Strep/10% FBS. After 16 h at 37°C/5% CO₂, the media was removed from the cells. Cells were then starved for 2 h in DMEM + 1% Pen/Strep. (A) Cells were pretreated with 5 ng/mL ultrapure LPS-RS, 10 µg/mL anti-hTLR4-IgG, or 10 µg/mL mouse IgG 1 control for an hour. Then cells were treated with 5 ng/mL ultrapure LPS-EB. Cells incubated with treatments in the incubator for 24 h. Afterward 20 µL of the cell supernatant was added to 180 µL QUANTI-Blue reagent. After incubation for 3 h at 37°C, the plate was read at 655 nM room temperature. Data are represented as mean (SD). After testing for homogeneity of variances, Tukey’s HSD was performed and adjusted (if applicable) (*p < 0.001). Comparisons shown are against LPS-EB (*p < 0.001). (B) Cells were pretreated with 5 ng/mL ultrapure LPS-RS, 10 µg/mL anti-hTLR4-IgG, or 10 µg/mL mouse IgG 1 control for an hour. Then cells were treated with 1 µg rAlt a 1. Cells incubated with treatments in the incubator for 24 h. Afterward, 20 µL of the cell supernatant was added to 180 µL QUANTI-Blue reagent. After incubation for 3 h at 37°C, the plate was read at 655 nM room temperature. Data are represented as mean (SD). After testing for homogeneity of variances, Tukey’s HSD was performed and adjusted (if applicable) (*p < 0.001). Comparisons shown are against Alt a 1 + IgG (*p < 0.001). (C) Cells were treated with 1 µg rAlt a 1. Data are represented as mean (SD). After testing for homogeneity of variances, Tukey’s HSD was performed and adjusted (if applicable) (*p < 0.001). Comparisons shown are against (A) LPS-EB, (B) Alt a 1 alone or Alt a 1 + IgG, and (C) Alt a 1 untreated null and hTLR2 cells and Alt a 1 treated null cells.