Fig. 1: LPS decreases microglial RhoA activity.

A Primary cortical microglia expressing the Raichu-RhoA biosensor exposed to 1 µg/ml LPS (n = 6 cells pooled across 3 independent cultures). Panels represent time-lapse FRET/CFP images coded according to the pseudocolor ramp. Graph (mean and SD) displays FRET/CFP ratio changes normalized at 0 min. *p < 0.05 (One-way ANOVA). B Primary cortical microglia expressing the Raichu-RhoA biosensor were exposed to different concentrations of LPS for 20 min (n = 10 cells pooled across 5 independent cultures). Graph (mean and SD) displays FRET/CFP ratio changes. The blue line shows untreated cells’ mean FRET/Donor ratio changes. The dashed line indicates the IC₅₀. C, Primary cortical microglia treated and non-treated (CT) with LPS (1 µg/ml) for 1 h (n = 10 independent cultures) and immunostained for GTP-RhoA. Graph (mean and SD) shows amounts of GTP-RhoA as mean fluorescent intensity (MFI). ****p < 0.0001 (Mann-Whitney test). D RhoA pull-down on lysates from primary cortical microglia treated and non-treated (CT) with LPS (1 µg/ml) for 1 h (n = 3 independent cultures). Graph displays mean with SD. **p < 0.01 (Mann-Whitney test). E HMC3 microglia expressing Raichu-RhoA biosensor treated and non-treated (CT) with 1 µg/ml LPS for 1 h (n = 100 cells per group from 10 independent experiments). Pseudocolor ramp represents min/max FRET/CFP ratios. Graph (mean and SD) displays FRET/CFP ratio changes. ****p < 0.0001 (unpaired t-test). F RhoA pull-down on lysates from HCM3 microglia treated and non-treated (CT) with LPS (1 µg/ml) for 1 h (n = 3 independent cultures). Graph displays mean with SD. *p < 0.05 (Mann-Whitney test). G Immunofluorescence images of GTP-RhoA and Iba-1 on cortical tissue sections from brain cortex of adult mice injected with saline or LPS (4 mg/Kg; 24 h). Graph (mean and SD) shows amounts of GTP-RhoA in Iba-1+ cells (n = 3 mice per group). *p < 0.001 (paired t-test). Scale bars: 10 µm (A, C, and G); 20 µm (E).