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. 2016 Jul 5;291(35):18058–18071. doi: 10.1074/jbc.M116.734186

FIGURE 4.

FIGURE 4.

KD of the shared BAF/PBAF core subunit SNF5. SNF5 was knocked down in hematopoietic progenitors and cells were differentiated into BMDMs as described under “Experimental Procedures.” A, mRNA levels of the Smarcb1 gene were analyzed in untreated BMDMs, as well as in cells transduced with control and specific shRNAs, as indicated. Results in cells transduced with shSmarcb1-2 and shSmarcb1-3 are shown as an average. Cells were either grown without LPS (blue, min LPS) or with LPS (yellow, plus LPS) for 1.5 h, and data were normalized to uninduced BMDMs. One-way ANOVA shows statistical significance between differently treated cells (p < 0.05), and a post hoc Tukey HSD test confirms statistical significance between untreated (UNT) (or shLuc-treated) and shSmarcb1-1- and shSmarcb1-2- or shSmarcb1-3-treated cells. *, p < 0.01. B, SNF5 protein was analyzed in the chromatin fractions of untreated BMDMs and of cells transduced with shSmarcb1-1. Western analysis shows loss of SNF5 and BRG1 in the SNF5 KD. RNA polymerase II (POLII) and histone H3 are shown as controls. Relative abundance of proteins when compared with untreated BMDMs is indicated. C, mRNA of Il12b (red) and Il1a (blue) in cells as described in A and grown in the presence of LPS for 1.5 h. One-way ANOVA shows statistical significance between differently treated cells (p < 0.05), and a post hoc Tukey HSD test confirms statistical significance between untreated and shSmarcb1-1- or shSmarcb2/3-treated cells for Il12b and for shSmarcb1-1-treated cells for Il1a. *, p < 0.01. D, mRNA of the macrophage markers Csf1r (blue) and Emr1 (orange) is shown in cells as in A grown in the absence of LPS. E and F, a BAF155 ChIP (E) and a SNF5 ChIP (F) were performed in untreated BMDMs grown in the absence (blue) or presence (yellow) of LPS for 1.5 h or in cells knocked down for SNF5 (shSmarcb1-1) and grown in the absence (green) or presence (red) of LPS. BAF155 binding to Il12b, Il1a, and control regions is shown as described in the legend for Fig. 1A. One-way ANOVA shows that differences between BMDMs and SNF5 KD cells are statistically significant (p < 0.05). A post hoc Fisher LSD test confirms that differences at the enhancers are statistically significant, whereas differences at control regions are not. Prom, promoter.