Figure 2.

PLA Detection of PRR Activation in Cells with Modulated PRR Expression Levels

(A) NIH/3T3 cells were transfected with or without TLR7 via electroporation. After 48 hr, cells were transfected with or without 2.5 μg luciferase mRNA by Lipofectamine 2000 (L2K). Cells were fixed after 24 hr with 1% paraformaldehyde, and PLA was performed between TLR7 and IRAK4. 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 one-way ANOVA with a Dunn’s multiple comparisons test, where n = 30 and ****p < 0.0001. 95% confidence intervals are shown in red. (B) RAW-Lucia ISG macrophages, WT, RIG-I KO, or MDA5 KO, were transfected with or without 2.5 μg luciferase mRNA by Lipofectamine 2000. Cells were fixed after 24 hr with ice-cold methanol, and PLA was performed between RIG-I and MAVS or MDA5 and MAVS. 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 one-way ANOVA with a Dunn’s multiple comparisons test, where n = 30 and *p < 0.035. 95% confidence intervals are shown in red. (C) RAW 264.7 macrophages were transfected with or without 100 nM TLR4 siRNA via Lipofectamine 2000. After 24 hr, cells were incubated with either 1 or 10 μg/mL LPS for TIRAP and TRAM interactions, respectively. After 30 min, cells were fixed with 1% paraformaldehyde, and PLA was performed between TLR4 and TIRAP and TLR4 and 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 one-way ANOVA with a Dunn’s multiple comparisons test, where n = 30 and ****p < 0.0001. 95% confidence intervals are shown in red.