Figure 3.

PLA Sensitively Detects PRR Activation Kinetics

(A) RAW 264.7 macrophages were incubated with the following agonists: 8 μg/mL imiquimod for TLR7, 10 μg/mL 5′ppp dsRNA for RIG-I, 0.1 μg/mL poly(I:C) for MDA5, 1 μg/mL LPS for TLR4-TIRAP, and 10 μg/mL LPS for TLR4-TRAM. Cells were fixed with 1% paraformaldehyde or methanol as previously indicated after 30 min or 6 hr. Also at 6 hr, agonists were removed and cells were incubated with media for 3 additional hours before fixing. PLA was performed between TLR7-IRAK4, RIG-I-MAVS, MDA5-MAVS, TLR4-TIRAP, and TLR4-TRAM. Representative images of PLA (white) and quantification of PLA are shown. Extended focus images are shown. Scale bars, 10 μm. Statistics were performed with a two-way ANOVA with a Tukey’s multiple comparisons test, where n = 30 and *p < 0.031, ****p < 0.0007, and ****p < 0.0001. 95% confidence intervals are shown in black. (B) A549 cells for TLR7-IRAK4, TLR4-TIRAP, and TLR4-TRAM PLAs were transfected with CellLight Early Endosomes-GFP-BacMam 2.0 for 16 hr prior to stimulation with 8 μg/mL imiquimod for TLR7 or 1 μg/mL LPS for TLR4-TIRAP and 10 μg/mL LPS for TLR4-TRAM for 30 min before fixation with 1% paraformaldehyde. RAW 264.7 macrophages for RIG-I-MAVS and MDA5-MAVS PLAs were incubated with MitoTracker for 15 min prior to stimulation with 0.1 μg/mL poly(I:C) for RIG-I or MDA5 for 30 min before fixation with methanol. PLA was performed between TLR7-IRAK4, RIG-I-MAVS, MDA5-MAVS, TLR4-TIRAP, and TLR4-TRAM. Representative images of PLA (red) and either endosomes (green) (TLR7-IRAK4, TLR4-TIRAP, and TLR4-TRAM) or mitochondria (green) (RIG-I-MAVS and MDA5-MAVS) are shown. Extended focus images are shown. Scale bars, 10 μm. (C and D) RAW 264.7 macrophages were incubated with the following agonists: 8 μg/mL imquimod, 10 μg/mL 5′ppp dsRNA, 0.1 μg/mL poly(I:C), and 1 or 10 μg/mL LPS. Supernatants were collected, and cells were fixed with 1% paraformaldehyde or methanol as previously indicated after 30 min or 6 or 24 hr. (C) An ELISA for TNF-α was performed on supernatants. Quantification is shown with SDs in black. Statistics were performed with a two-way ANOVA with a Tukey’s multiple comparisons test, where n = 2 and *p < 0.007 and ****p < 0.0001. (D) PLA was performed at the 30-min time point for TLR7-IRAK4, RIG-I-MAVS, MDA5-MAVS, TLR4-TIRAP, and TLR4-TRAM. Quantification is shown, with 95% confidence intervals in black. Statistics were performed with a two-way ANOVA and a Tukey’s multiple comparisons test, where n = 30 and *p < 0.01, **p < 0.007, and ****p < 0.0001.