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. 2024 Jun 28;20(8):5511–5527. doi: 10.1002/alz.14047

FIGURE 3.

FIGURE 3

PKCδ is highly expressed in microglia and modulates microglia‐mediated neuroinflammation. (A) Western blot analysis of the expression of PKCδ and cellular markers including NeuN (for neuron), HS1 (for microglia), and GFAP (for astrocyte) in mouse primary cellular cultures. (B) Representative images of immuno‐stained PKCδ in MAP2+, GFAP+, and IBA1+ cells in WT and APPswe/PS1dE9 mouse brain. (C) Quantification of PKCδ+MAP2+, PKCδ+GFAP+, and PKCδ+IBA1+ cells in the brain of APP/PS1 mice relative to WT mice. n = 4 mice/group, two‐way ANOVA with Sidak multiple comparisons test. (D) Quantification of fluorescence intensity of PKCδ in neuronal (MAP2+), astrocytic (GFAP+), and microglial (IBA1+) cells in APPswe/PS1dE9 brains relative to WT controls. n = 4 mice/group, two‐way ANOVA with Sidak multiple comparisons test. (E) Western blot analysis of secreted and intracellular PKCδ in primary microglial culture in the presence or absence of Aβ oligomers. n = 5 biological replicates, unpaired t‐test. (F) Western blot analysis of secreted and intracellular PKCδ in primary microglial culture with or without LPS stimulation. n = 5 biological replicates, unpaired t‐test. (G–J) Primary microglia were transduced with lentiviruses for 5 days and then subjected to the analyses of western blot, qRT‐PCR, or RNA‐seq. (G, H) Western blot analysis of PKCδ expression. n = 5 biological replicates, unpaired t‐test. (I) qRT‐PCR analysis of IL‐1β, IL6, and TNFα expression. n = 5 biological replicates, unpaired t‐test. (J) Volcano plot indicating differentially expressed genes revealed by RNA‐seq; colored plots represent significantly downregulated (green) and upregulated (red) genes. Log10 Q value (y‐axis) and FC (log2FC, PKCδ vs Ctrl, x‐axis) are shown. Significance cutoffs were set to p < 0.05, FC > 1.2. (K) GO pathway analysis of differentially regulated genes. (L) KEGG pathway analysis of differentially regulated genes. (M) Heatmap depicting NF‐κB ‐signal‐pathway‐related genes. (N) qRT‐PCR analysis of Ccl5, Cxcl3, Cxcr1, Tlr4, Cd40, and Tnfs40 expression, n = 5 biological replicates, unpaired t‐test. Scale bar = 20 µm. Data represent mean ± SD; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant. Aβ, amyloid beta; APP, amyloid precursor protein; IL‐1β, interleukin 1 beta; KEGG, Kyoto Encyclopedia of Genes and Genome; NF‐κB: nuclear factor‐kappa B; PKCδ, protein kinase C delta; qRT‐PCR, quantitative real‐time PCR.