Figure 5.

K306 selectively increases microglial degradation of membrane-containing phagolysosomal cargo

BV2 pretreated with 10μM K306 for 1 h was incubated with the indicated cargo for 1 h and degradation of the cargo was detected by either fluorescence microscopy (A) or flow cytometry (C and D).

(A) Image of TdTomato-labeled synaptosomes phagocytosed by BV2 cells at time 0 (T0) for uptake and 6 h (T6H) after uptake for DMSO control or K306 treatment.

(B) Quantification of area covered by synaptosomes and mean intensity for synaptosome per cell.

(C) Dead neuron uptake (T0) and degradation (T6H) evaluated by flow cytometry as mean fluorescence intensity of PI-labeled engulfed dead neurons for BV2 cells treated with DMSO or K306 (10μM).

(D) Flow cytometry detection of Aβ42-FITC uptake (T0) and degradation at 2 and 6 h (T2H, T6H). (Data are representative of four independent experiments in (A and B) or three independent experiments in (C, D). Bars indicate mean ±SEM. The significance of engulfment or degradation for K306 vs. vehicle was assessed by unpaired Student’s T-test (C) or via a two-way ANOVA(B, D) with Tukey correction for multiple comparisons. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05).

(E) Residual Aβ38, Aβ40, and Aβ42 levels from HeLa swAPP-conditioned medium, after 6 h incubation with BV2 cells treated with K306 or DMSO control, normalized to DMSO and to cell viability. Bars indicate mean ± SEM from three independent experiments.