Fig 2: Cisplatin increases the expression of M1, M2, and Iba1 markers in the auditory nerve. (A) Macrophage marker expression in different groups. On day 8 post-cisplatin injection, there was a marked increase in the expression of M1 markers (CD68 and CD86), M2 markers (CD206 and CD163), and Iba1. This increased expression was temporary and showed recovery on day 15. This increased expression suggested a temporary auditory nerve inflammation. There was no difference between the expressed markers in the control group. (B) Statistical analysis of the ratio of each marker expression in F4/80+ macrophages. In all the markers, there was significant increase in the ratio of expression on day 8 in cisplatin-injected mice (n = 10) compared to that of the day 8 control (n = 8), day 0 pre-injection (n = 6), and day 15 cisplatin-injected mice (n = 8). ****P < 0.0001, ns, not significant by two-way ANOVA with Bonferroni post hoc test. The error bars represent standard deviation from the mean.

Fig 3: Characterization of macrophage phenotype across mouse strains after exposure to polarizing stimuli. Bone marrow derived macrophages from each strain were polarized with vehicle (PBS), LPS (1 ng/ml) or IL-13 (10 ng/ml). After 3 days of exposure to each stimulus, total RNA was isolated for further assays. (A) Polarization in response to LPS (M1) or IL-13 (M2) was evaluated by assessing the level of Cd86 and Arg1 mRNA with quantitative PCR (QPCR) normalized to β-actin mRNA. (B) The expression of genes related to M2 polarization was evaluated in macrophages treated with vehicle or IL-13 using the NanoString nCounter Gene Expression Assay and a custom code set. Unsupervised hierarchical clustering of M2 related genes was performed (left panel). (C) mRNA expression of Mrc1, Il10, Ccl2 and Ccl17 in polarized macrophages was confirmed by QPCR. (D) The concentrations of IL-10, IL-13, CCL2 and CCL17 in supernatants collected from polarized macrophages were determined with ELISA. Veh: PBS, BL6J: C57BL6/J, HeN: C3H/HeN. Columns: mean, error bars: +SD, *p<0.05 for comparison to the corresponding macrophages treated with vehicle. §p<0.05 for comparison to C57L macrophages exposed to IL-13 by ANOVA with Tukey’s correction.

Fig 4: | EPZ6438 decreased the infiltration proportion of M2 macrophages but GSK126 increased it in vivo. (A) The statistical graph of flow cytometry on CD11b+ F4/80+ cell ratio in CD45+ cells representing total macrophages in the tumor microenvironment. (B) The ratio of CD86+ cells in CD11b+ F4/80+ cells representing M1 macrophages. (C) The ratio of CD206+ cells in CD11b+ F4/80+ cells representing M2 macrophages. (D) Opal multiplex immunofluorescence staining identified the proportion and location of M1 and M2 macrophages in the tumor microenvironment. Individual staining images of DAPI, F4/80, CD206, CD86, separately (left) and the merged images (right). (E–G) The quantitative analysis regarding the Opal multiplex immunofluorescence staining as shown above. (H) The level of H3K27me3 on the promoter of STAT3 was analyzed by the ChIP-qPCR experiment. The level of H3K27me3 was decreased after GSK126 and EPZ6438 treatment. The experimental results were normalized to a 2% input sample group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance.

Fig 5: BRD9 degrader mitigates ZOL-related ONJ after tooth extraction.a Schematic illustrating the protocol used to establish ZOL-related ONJ model and dBRD9 degrader treatment. b μCT imaging of the maxillary bone from mice in dBRD9 degrader treated group and the control group. Yellow arrow 1 indicates severe periosteal reaction. Yellow arrow 2 indicates hyper-osseous maxillary sinus floor. Yellow arrow 3 indicates osteosclerotic patterns and sequestrum. Asterisk indicates insufficient amount of bone. Scale bar, 1 mm. c The incidence rate of osteonecrosis and periosteal reaction of the maxillary bone from mice in dBRD9 degrader treated group and the control group. n = 5. d H&E staining of the periodontal and alveolar bone area from mice in BRD9 degrader treated group and the control group. Colored boxes in the left are shown at higher magnification in right. Black dashed lines outline the boundary of the healed extraction sockets. Asterisk indicates necrotic alveolar bone with empty lacunae. Scale bar, 200 μm. e TRAP staining and f quantification of the periodontal and alveolar bone area from mice in BRD9 degrader treated group and the control group. Colored boxes in the left are shown at higher magnification in right. Arrows point to TRAP-positive osteoclasts. Asterisk indicates no signal. Scale bar, 200 μm. n = 5. Immunofluorescence staining of g iNOS (red), h TNF-a (red), i CD86 (red), k STAT1 (red) of the periodontal and alveolar bone area from mice in BRD9 degrader treated group and the control group. Colored boxes in the left are shown at higher magnification in right. Blue dashed lines outline the boundary of the healed extraction sockets. Arrows indicates positive signal. Scale bar, 200 μm. j The mRNA expression of Brd9 in ZOL or vector-treated BMDMs during LPS-induced macrophage activation and RANKL-induced osteoclastogenesis at indicated timepoint. n = 3 biologically independent samples. All data in this figure are represented as mean ± SD. Two-tailed Student’s t-test for (f) and (j). All experiments were performed in triplicates unless otherwise stated. Source data are provided in the Source data file.