Fig. 3

HG enhanced AKAP8L and mTORC1 interaction, and both si-AKAP8L and rapamycin mitigate HG-induced autophagy disorders in microglia. Co-immunoprecipitation of FLAG-tagged AKAP8L and HA-tagged Raptor (a), and vice versa (b), as well as the interaction between endogenous AKAP8L and Raptor (c). Proximity ligation assay reveals increased AKAP8L-Raptor interactions in BV2 cells (d) and primary microglia (e). Scale bar: 20 μm. (f) Representative blots of autophagy-related proteins. Statistical graphs of protein expression for p-mTOR/mTOR (g), p-p70S6K/p70S6K (h), p-ULK1/ULK1 (i), Beclin-1 (j), p62 (k), and LC3 II/LC3 I (l). (m) Immunofluorescence staining of microglial cells transfected with mRFP-GFP-LC3 adenovirus, showing punctate LC3 distribution. Scale bar: 10 μm. (n) Electron microscopy images of microglia reveal HG-induced autophagy alterations. Scale bar: 500 nm. (o) LC3 puncta numbers per cell. (p) Quantification of autophagosome (GFP + RFP + yellow puncta) and autolysosome (red puncta) numbers in microglia. Data are Mean ± SD (n = 5). P-values are determined using unpaired Student’s t-tests for two-group comparisons and one-way ANOVA with Tukey’s post hoc test for multiple comparisons. **p < 0.01 compared to the Control group. ^#p < 0.05 and ^##p < 0.01 compared to the STZ group