Fig 2: Diet-induced macrophage changes in MAT and EAT.(A) F4/80 expression (qPCR relative to GAPDH) in MAT and EAT (n = 4–8). (B) MCP-1 expression (qPCR relative to GAPDH) in MAT and EAT (n = 4–8). (C) Changes in MCP-1 protein detected by ELISA in extracts of adipose tissues and expressed as ratios of HMF diet over control diet adipose tissue levels in EAT and MAT. (n = 4–8) (D) Typical flow cytometry pattern of the SVF of the MAT showing (a) F4/80Int CD206+ and (b) F4/80HI CD206- macrophage populations. (E) Changes in the F4/80Int CD206+ macrophage population expressed as ratios of HMF diet over control diet adipose tissue cells/gm in EAT and MAT (n = 6). (F) Representative flow cytometry of the F4/80HI CD206- macrophage population in MAT showing CD11b expression with control and HMF diets; and graph of the percentage of these cells in the total macrophage population within the stromavascular fraction of MAT from control and HMF diets. (G) Changes in the F4/80HI CD206- CD11bHi macrophages expressed as ratios of cells/g in HMF diet over control diet EAT and MAT.

Fig 3: Macrophage-specific Smad3 is a key regulator of MMT in NSCLC. Smad3 deletion significantly suppresses MMT (a-SMA+ F4/80+) compared to Smad3-WT mice: A) visualized by immunofluorescence and B) quantified by flow cytometric analysis (** p < 0.01 vs WT, n = 4, t-test). C) Immunofluorescence and quantification shows that Smad3 deletion in adoptively transferred BMDM significantly suppresses the level of MMTs and a-SMA+ CAFs in the LLC tumor of the S3KO-BMDM group compared with S3WT-BMDM group (***p < 0.001 vs control, ###p < 0.001 vs S3WT-BMDM, one-way ANOVA, n = 4). D) Live bioluminescence imaging, and tumor volume shows macrophage-specific Smad3 blockade significantly inhibits the LLC-luc cancer progression in S3KO-BMDM group compared with the S3WT-BMDM group (***p < 0.001 vs control, ###p < 0.001 vs S3WT-BMDM, one-way ANOVA, n = 4). Immunofluorescent shows that Smad3 inhibition by specific inhibitor SIS3 significantly suppresses E) the level of MMTs and F) tumor growth in the 5 mg kg-1 SIS3 treatment group compared with the solvent control group (***p < 0.001 vs control, **p < 0.01 vs control, one-way ANOVA, n = 4). Scale bar, 50 µm.

Fig 4: Characterization of MM-MFs in a murine myeloma model.A Flow cytometry showed murine BM-derived MFs and MM-MFs were positive of F4/80 and CD11b staining. B Mean fluorescent intensity (MFI) of flow cytometry indicated in vitro cultured MM-MFs expressed higher CD206 than MFs. C In CFSE proliferation assay, MM-MFs showed higher cell proliferation capacity than MFs. D Cell cycle analysis showed that MM-MFs had more S and G2/M population, relative to MFs. E ELISA assay detected the concentration of VEGF in culture supernatant of murine BM-derived M?s and MM-M?s. Statistical significance was determined by two-tailed Student t-test between M?s and MM-M?s, **P < 0.01. F Culture supernatant of murine MM-M?s had higher nitric oxide concentration, relative to MFs. Statistical significance was determined by two-tailed Student t-test between M?s and MM-M?s, *P < 0.05. G 5 T murine myeloma model was used for identifying in vivo MM-MF features, IVIS photos (left) and CD138 staining (right panel) of BM cells illustrated myeloma tumor burden. Results from 3 representative mice from 7 injected mice are shown. H MFs from myeloma-bearing mice expressed higher CD206 than those from health mice.

Fig 5: G9a knockdown can promote the polarization of pulmonary macrophages from M1 pro-inflammatory phenotype to M2 anti-inflammatory phenotype. G9a lentivirus interference vector was injected into a Streptococcus pneumonia-induced pneumonia mouse model. (A) Surface markers of M1 macrophages CD86 and M2 macrophages CD206 were detected by flow cytometry. Firstly, cell debris (black dots out of frame) were removed by FSC and SSC, then living (blue dots) and dead (red dots) cells were distinguished by PI labeling. Finally, M1 and M2 macrophages were sorted out by F4/80 (blue dots), CD86, and CD206. The percentage of all cells refers to the percentage of living cells; (B) the expression of M1 macrophage markers iNOS and M2 macrophage markers Arg-1 were detected by RT-qPCR (N=5 in each group). The data are expressed as mean ± SD; and one-way ANOVA and Tukey’s post hoc test was used for comparison among multiple groups. *, P<0.05. RT-qPCR, reverse transcription quantitative polymerase chain reaction. FSC, forward scatter; SSC, side scatter; PI, propidium iodide; iNOS, inducible nitric oxide synthase; Arg-1, Arginase-1; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SD, standard deviation; ANOVA, analysis of variance; ns, no significance.