Figure 3.
Mechanism of action of A11. (a) EMSA using 20 fmol of biotinylated DNA probes, or 4 pmol non-biotinylated “cold” probes as negative control. The “Probe shift” condition of lane 2 represents the presence of lysate and biotinylated probe, whereas lane 1 lacks the lysate and lane 3 has a cold probe added in excess. Lanes 4–15 are identical to probe shift but with either DMSO or different concentrations of A11 added. Probes contained no PU.1 motif (Epstein-Barr), a single PU.1 motif (λB; sequence shown in Fig. 1 d), a double PU.1 motif (2XλB), or a previously published ETS-IRF motif (Eisenbeis et al., 1993). EMSA was repeated in four separate batches of experiments. (b) Quantification of band intensity versus DMSO, fitted to Hill’s equation. (c) Shift-western: EMSA Western blots were generated under native or SDS-denatured conditions and stained with a PU.1 antibody. Lane 1 shows only 0.5 ng of recombinant PU.1 protein. Lane 2 shows only probe. Lane 3 shows lysate and PU.1 protein. Lane 4 shows lysate and probe added. Lanes 5–15 are identical to lane 4 but with either DMSO or A11 added at different concentrations. Results in this panel were repeated in three separate batches of experiments. (d) Quantification of band intensity versus DMSO (indicated by magenta arrow for non-denatured sample), fitted to Hill’s equation. (e) Pulldown of the biotinylated λB motif using streptavidin beads, followed by SDS-denaturing before gel loading and then Western blotting with antibodies for various binding partners of PU.1. Lane 1 sample contains 5% input (iMGL lysate) only. Lane 2 sample contains iMGL lysate without biotinylated probe. Lane 3 contains biotinylated probe but no lysate. Lanes 4 and 5 contain biotinylated probe and iMGL lysates, in addition to DMSO and 500 µM A11. Band intensity quantification to the right, with DMSO set as 1. Student’s unpaired, two-sided t test. Bars are mean ± SEM; each data point represents a well. Results in this panel were repeated in three separate batches of experiments. (f) ChIP using a MECP2 antibody and HDAC1 antibody, followed by qPCR for several PU.1 target genes. (1) and (2) after the gene name denotes that two separate primer pairs used. Student’s unpaired, two-sided t test; ***P ≤ 0.001, *P ≤ 0.05, n = 4 for all conditions. Bars are mean ± SEM; each data point represents a well. (g) Volcano plot of DEGs (Padj ≤ 0.05; calculated with apeglm (bulk RNA sequencing) of mouse microglia isolated by FACS from mice treated with A11 (0.3 mg/kg intraperitoneally, every day, for 2 wk) compared to treatment with vehicle (1% DMSO in DPBS). (h) Motif analysis of the DEGs in g using HOMER software ± 2,000 bp from the transcription start site. Padj represents Benjamini corrected q-values. (i) Effect of A11 on the 2060 PU.1 target genes upregulated in CK-p25 mice (from Fig. 1 b). (j) Gene ontology analysis of the downregulated genes from panel i with (Padj ≤ 0.05, −1 > log2(fold change) > 1) using Toppgene, accessed March 4, 2023. Padj represents Bonferroni corrected P values. (k) ChIP using a HDAC1 antibody, followed by qPCR for several PU.1 target genes. (1) and (2) after the gene name denotes that two separate primer pairs are used. Student’s unpaired, two-sided t test; *P ≤ 0.05, n = 4 for all conditions. Bars are mean ± SEM; each data point represents a well. Source data are available for this figure: SourceData F3.

Mechanism of action of A11. (a) EMSA using 20 fmol of biotinylated DNA probes, or 4 pmol non-biotinylated “cold” probes as negative control. The “Probe shift” condition of lane 2 represents the presence of lysate and biotinylated probe, whereas lane 1 lacks the lysate and lane 3 has a cold probe added in excess. Lanes 4–15 are identical to probe shift but with either DMSO or different concentrations of A11 added. Probes contained no PU.1 motif (Epstein-Barr), a single PU.1 motif (λB; sequence shown in Fig. 1 d), a double PU.1 motif (2XλB), or a previously published ETS-IRF motif (Eisenbeis et al., 1993). EMSA was repeated in four separate batches of experiments. (b) Quantification of band intensity versus DMSO, fitted to Hill’s equation. (c) Shift-western: EMSA Western blots were generated under native or SDS-denatured conditions and stained with a PU.1 antibody. Lane 1 shows only 0.5 ng of recombinant PU.1 protein. Lane 2 shows only probe. Lane 3 shows lysate and PU.1 protein. Lane 4 shows lysate and probe added. Lanes 5–15 are identical to lane 4 but with either DMSO or A11 added at different concentrations. Results in this panel were repeated in three separate batches of experiments. (d) Quantification of band intensity versus DMSO (indicated by magenta arrow for non-denatured sample), fitted to Hill’s equation. (e) Pulldown of the biotinylated λB motif using streptavidin beads, followed by SDS-denaturing before gel loading and then Western blotting with antibodies for various binding partners of PU.1. Lane 1 sample contains 5% input (iMGL lysate) only. Lane 2 sample contains iMGL lysate without biotinylated probe. Lane 3 contains biotinylated probe but no lysate. Lanes 4 and 5 contain biotinylated probe and iMGL lysates, in addition to DMSO and 500 µM A11. Band intensity quantification to the right, with DMSO set as 1. Student’s unpaired, two-sided t test. Bars are mean ± SEM; each data point represents a well. Results in this panel were repeated in three separate batches of experiments. (f) ChIP using a MECP2 antibody and HDAC1 antibody, followed by qPCR for several PU.1 target genes. (1) and (2) after the gene name denotes that two separate primer pairs used. Student’s unpaired, two-sided t test; ***P ≤ 0.001, *P ≤ 0.05, n = 4 for all conditions. Bars are mean ± SEM; each data point represents a well. (g) Volcano plot of DEGs (Padj ≤ 0.05; calculated with apeglm (bulk RNA sequencing) of mouse microglia isolated by FACS from mice treated with A11 (0.3 mg/kg intraperitoneally, every day, for 2 wk) compared to treatment with vehicle (1% DMSO in DPBS). (h) Motif analysis of the DEGs in g using HOMER software ± 2,000 bp from the transcription start site. Padj represents Benjamini corrected q-values. (i) Effect of A11 on the 2060 PU.1 target genes upregulated in CK-p25 mice (from Fig. 1 b). (j) Gene ontology analysis of the downregulated genes from panel i with (Padj ≤ 0.05, −1 > log2(fold change) > 1) using Toppgene, accessed March 4, 2023. Padj represents Bonferroni corrected P values. (k) ChIP using a HDAC1 antibody, followed by qPCR for several PU.1 target genes. (1) and (2) after the gene name denotes that two separate primer pairs are used. Student’s unpaired, two-sided t test; *P ≤ 0.05, n = 4 for all conditions. Bars are mean ± SEM; each data point represents a well. Source data are available for this figure: SourceData F3.

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