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Fig. 4 | Molecular Neurodegeneration

Fig. 4

From: PU.1 regulates Alzheimer’s disease-associated genes in primary human microglia

Fig. 4

Confirmation of microarray analysis in isolated human brain microglia. a Isolated microglia cultures transfected for 7 days with 50 nM PU.1 siRNA show no change in overall cell number (p > 0.05), (b) no change in the percentage of microglial cells (p > 0.05), (c) efficient knockdown of PU.1 (p < 0.001), (d, g) reduced expression of DAP12 (p < 0.001) and HLA-DR, DP, DQ (p < 0.01) but not CD45 (p > 0.05), (e) non-significant induction in roundness factor (p > 0.0.5), and (f) reduced elongation factor (p < 0.001) compared to a scrambled siRNA control, n = 3–5 independent microglial cultures, scale bar = 100 μm. h qRT-PCR validation of selected genes in isolated microglia cultures transfected with 50 nM PU.1 siRNA versus control siRNA for 7 days, colour coded by their significance from microarray analysis, (i) displayed a significant correlation to changes observed in mixed glial cultures (R2 = 0.55), n = 3 independent microglial cultures. Data is displayed as fold change of mRNA genes in PU.1 siRNA treated cultures relative to control siRNA samples as determined by the 2^-ΔΔCt method. Cytokine secretions from isolated microglia cultures transfected with 50 nM PU.1 siRNA versus control siRNA for 6 days followed by stimulation with vehicle or 10 ng/mL LPS for a further 24 h demonstrated no effect of PU.1 silencing on LPS-induced (j) IL-1β (p > 0.05), (k) TNFα (p > 0.05), (l) IL-6 (p > 0.05), (m) MCP-1 (p > 0.05) but a reduction in (n) IL-8 (p < 0.001). Please note that this is one representative result of three independent experiments. NS = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, Students t test

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