Supplementary Figure 8: Analysis of phagocytosis in BV2 microglial cells.
From: A common haplotype lowers PU.1 expression in myeloid cells and delays onset of Alzheimer's disease
(a) Flow cytometry histograms of BV2 cells transfected with pcDNA3 (pcDNA) or pcDNA3-FLAG-PU.1 (FLAG-PU.1) with pCMV-GFP for overexpression and scrambled shRNA (shSCR) or shRNA targeting PU.1 (shA, shB and shD) in pGFP-V-RS vector for knock-down of PU.1 after 3 hours of incubation with pHrodo-labeled zymosan. Cells were gated on GFP+ population. (b) Flow cytometry analysis of the number of gated cells in a presented as mean ± s.d., pcDNA 67.03 ± 6.883, pcDNA + 1 μM Cyt 15.64 ± 16.24, FLAG-PU.1 82.71 ± 4.74, shSCR 77.17 ± 3.115, shA 48.63 ± 2.285, shB 28.92 ± 2.495, shD 22.76 ± 1.595. pcDNA vs pcDNA + 1 μM Cyt P < 0.0001, pcDNA vs FLAG-PU.1 P = 0.0306, shSCR vs shA P = 0.0002, shSCR vs shB P < 0.0001, shSCR vs shD P < 0.0001. F(6,13) = 58.68, n = 3. (c) Flow cytometry analysis of the geometric mean fluorescent pHrodo intensity in a presented as mean ± s.d., pcDNA 10952 ± 2206, pcDNA + 1 μM Cyt 1533 ± 47, FLAG-PU.1 15226 ± 2701, shSCR 13129 ± 4617, shA 9937 ± 2168, shB 8872 ± 2019, shD 8754 ± 1856. pcDNA vs pcDNA + 1 μM Cyt P = 0.0092. F(6,13) = 6.228, n = 3. (d) Flow cytometry histograms of BV2 cells transfected as in (a) and gated on GFP- populations. (e) Flow cytometry analysis of number of gated cells in d presented as mean ± s.d., pcDNA 63.92 ± 6.575, pcDNA + 1 μM Cyt 14.21 ± 13.66, FLAG-PU.1 67.54 ± 4.826, shSCR 68.31 ± 5.784, shA 67.27 ± 4.144, shB 65.19 ± 4.268, shD 60.3 ± 2.181. pcDNA vs pcDNA + 1 μM Cyt P < 0.0001. F(6,13) = 22.53, n = 3. (f) Flow cytometry analysis of geometric mean fluorescent pHrodo intensity in d presented as mean ± s.d., pcDNA 9186 ± 2863, pcDNA + 1 μM Cyt 1545 ± 147, FLAG-PU.1 9931 ± 2458, shSCR 9849 ± 3012, shA 10903 ± 2949, shB 10912 ± 2494, shD 10934 ± 2685. pcDNA vs pcDNA + 1 μM Cyt P = 0.0367. F(6,13) = 3.473, n = 3. (g) Phagocytic index of BV2 GFP- cells analyzed in (e) and (f) presented as mean ± s.d., pcDNA 0.5954 ± 0.2223, pcDNA + 1 μM Cyt 0.0209 ± 0.0189, FLAG-PU.1 0.6745 ± 0.188, shSCR 0.6765 ± 0.2274, shA 0.7382 ± 0.2255, shB 0.7131 ± 0.1742, shD 0.6612 ± 0.1748. pcDNA vs pcDNA + 1 μM Cyt P = 0.0331. F(6,13) = 3.53, n = 3. Cytochalasin D (Cyt) treatment was used as a negative control. * P < 0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA with Sidak’s post hoc multiple comparisons test between selected groups.
