Fig. 6
From: PU.1 regulates Alzheimer’s disease-associated genes in primary human microglia
High throughput drug screening identified vorinostat as an effective inhibitor of PU.1 that partially mimics the effects of PU.1 silencing. a High throughput drug screening using a drug library containing 1280 FDA-approved compounds at 10 μM for 48 h in mixed glial cultures. ICC and automated image analysis identified the HDAC inhibitor vorinostat as a candidate drug for PU.1 reduction. Please note that this screening experiment was performed using one biological sample. b Confirmation of the PU.1 inhibitory effect (p < 0.001) of 10 μM vorinostat for 48 h in additional mixed glial cultures analysed by ICC and automated image analysis and (c) a mild reduction (p < 0.05) in total cell number (c), n = 2. d ICC analysis demonstrating loss of nuclear PU.1 expression with vorinostat treatment in CD45+ microglia, scale bar = 100 μm. Isolated microglia cultures treated for 24 h with 10 μM vorinostat show (e) a reduction in cell number (p < 0.001), (f) no change in the percentage of microglial cells (p > 0.05), (g, k) efficient knockdown of PU.1 (p < 0.001), (h, k) reduced expression of DAP12 (p < 0.01) but not HLA-DR, DP, DQ (p > 0.05) or CD45 (p > 0.05), and no change in (i) roundness factor or (j) elongation factor compared to vehicle treatment, n = 3–5, scale bar = 100 μm. (l, m) Determination of gene changes following a 24 h treatment with 10 μM vorinostat demonstrated a modest correlation (R2 = 0.47) with genes altered by PU.1 silencing in isolated microglia cultures, n = 3 independent microglial cultures. Data is displayed as fold change of mRNA genes in vorinostat treated cultures relative to vehicle treated samples as determined by the 2^-ΔΔCt method. NS = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, Students t test