Fig 2: Phenelzine treatment polarizes macrophages in the tumor microenvironment toward an M1 phenotype. (A) Treatment regime using the BALB/c 4T1 breast cancer model. (B) Tumor volumes of mice treated with vehicle control, Abraxane, Phenelzine or PD1 (n = 4/5). (C) Flow cytometry for total macrophages, inflammatory macrophages, and M2-like macrophages in the TME. *p < 0.05, Mann–Whitney t-test (n = 4/5). Representative images of (D) M1 and (E) M2 staining of FFPE tumor tissues in 4T1 mouse model. (F) Sections of primary 4T1 tumors were fixed and IF microscopy performed probing with M1 focused primary antibodies to F4/80, iNOS, CD86, and PDL1 with DAPI (green = F4/80 red = iNOS, yellow = CD86, cyan = PDL1, blue = DAPI). The population % of F4/80 cells positive for iNOS, CD86 and PDL1 was measured using ASI's mIF system. Representative images for each dataset are shown. Graphs plots represent the % population (n = 500 cells profiled per a group, n = 4 mice). (G) Section of primary 4T1 tumors were fixed and IF microscopy performed probing with M2 focused primary antibodies to F4/80, EGR2, CD206, and PDL2 with DAPI (green = F4/80 red = EGR2, yellow = CD206, cyan = PDL2, blue = DAPI). The population % of F4/80 cells positive for EGR2, CD206, and PDL2 was measured using ASI's mIF system. Representative images for each dataset are shown. Graphs plots represent the % population (n = 500 cells profiled per a group, n = 4 mice).

Fig 3: Embelin inhibits the activation of M1 macrophages in the LPS mouse model. A) M1 marker (iNOS) and M2 markers (CD163 and CD206) were detected using IHC. B) KIM-1 (purple), CD206 (red), iNOS (green), and DAPI (blue) are illustrated through multiplex immunofluorescence. All fields were captured using confocal microscope using a 60 × oil immersion lens. The images shown are representative of three independent experiments. The scale bars shown are 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 4: Platelets, macrophage subsets, and T cell subsets in endometriotic lesions of the four groups of mice in Experiment 2. (A) Representative immunohistochemical staining of CD41, CD68, CD163, iNOS, TSLP, FOXP3, T-bet, GATA-3, and RORγT in the four groups. All magnifications were ×400. Scale bar = 50 µm. (B) Boxplots showing the density of CD41+ (platelet), CD68+ (macrophage), CD163+ (M2), and iNOS+ (M1), FOXP3+ (Treg), T-bet+ (Th1), GATA-3+ (Th2), RORγT+(Th17) cells, and the staining level of TSLP. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not statistically significant (p > 0.05) for the difference between the testing group and the Control group (by Wilcoxon’s test). n = 8 for each group. CT, Control group; PD, platelet depletion; TD, Treg depletion; JD, joint (simultaneous) depletion of platelet and Treg; TSLP, thymic stromal lymphopoietin.

Fig 5: SOCS1-siRNA reversed BBR-induced effects on iNOS expression and proinflammatory factor releases. The cells were divided into five groups, including the normal cultured control group, 5 μM Aβ exposure group, BBR treatment group (medium containing 5 μM BBR and 5 μM Aβ), SOCS1 treatment group (microglial cells were treated with SOCS1-siRNA for 6 h and then exposed to the medium containing 5 μM BBR and 5 μM Aβ), and scrambled- (SC-) siRNA treatment group (microglial cells were treated with SC-siRNA for 6 h and then exposed to the medium containing 5 μM BBR and 5 μM Aβ); then, after 24 h treatment, iNOS expression was observed by using immunocytochemistry staining and Western blot, and inflammatory factors were assessed by using ELISA. (a) Immunocytochemistry staining of microglial iNOS expression. (b) Western blot result of iNOS expression (n = 4). (c–e) TNF-α, IL-1β, and IL-6 concentrations in the medium (n = 8). Results are expressed as means ± SD. Bar = 10 μm; ∗P < 0.05; NS: no significance.