Fig 2: APG-115 suppresses alternative M2 macrophages polarization in vitro and increases M1 macrophages in vivo through activation of p53 pathway. a BMDMs were generated under the treatment with m-CSF for 7 days and then treated with IL-4 (20 ng/mL) to induce alternative macrophage polarization (M2) for 24 h in the absence or presence of APG-115. Cells were then harvested for detection of M2 macrophages (CD206+MHC-IIlow) by flow cytometry. b the mRNA expression levels of Arg-1 and Retnla in the above BMDMs induced by the treatment with IL-4 (20 ng/mL) with or without APG-115 were analyzed by RT-qPCR. Duplicated samples were tested. c Western blot analysis of p53, p21, c-Myc and c-Maf total proteins in BMDMs treated with IL-4 (20 ng/mL) with or without APG-115 (1 μM) for 0, 4, or 24 h, or sequentially treated with IL-4 and then APG-115 for 24 h each (24 h + 24 h). d Quantification of C. 0 (black bars), 4 (blue bars), or 24 h (green bars), or sequentially treated with each agent for 24 h (24 h + 24 h, red bars). e naïve BALB/c mice were treated with APG-115 (10 mg/kg, Q2D × 2 doses; n = 5). Two days after the last dose, spleens were collected, dissociated into single-cell suspensions, and stained with macrophage markers for flow cytometry analysis. Macrophages were defined as CD11b+ F4/80hi, and further analyzed for M1 macrophages by expression of MHC-II. Pooled data of percentages of macrophages gated on CD45+CD3− live cells (f) and percentages of M1 macrophages gated on macrophages (g) from five mice were plotted

Fig 3: Robust antitumor immune responses triggered by IPI549@HMP-augmented RT.a–f Representative flow cytometric analysis and relative quantification of CTLs (CD8+CD3+CD45+) (a, b), MDSCs (CD11b+Gr-1+CD45+) (c, d) and TAM-M2 (CD206hiCD11b+F4/80+CD45+) (e, f). g, h Quantification by flow cytometry of CD8/Treg (g) and M1/M2 ratios (h). i Representative polychromatic immunofluorescent staining images of tumors from three biologically independent samples showing DAPI (blue), CD206+ (green), Foxp3+ (orange) and CD8+ (red) cells infiltration for Control and IPI549@HMP + RT groups. j, k Cytokine levels of TNF-α (j) and IFN-γ (k) in the serum after varied treatments. G1, Control; G2, IPI549@HMP; G3, RT; G4, HMP + RT; G5, IPI549@HMP + RT. RT, radiotherapy; CTLs, cytotoxic T lymphocytes; Tregs, regulatory T cells; MDSCs, myeloid-derived suppressor cells; TAMs-M2, M2-like macrophages; TAMs-M1, M1-like macrophages. Data were expressed as means ± SD (n = 3 biologically independent samples in b, d, f, g, h, j, and k). Statistical difference was calculated using two-tailed unpaired student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.

Fig 4: House dust mite exposure induces airway eosinophilic and T helper 2-associated inflammation. (A) Flow cytometry analysis of eosinophils in the lung. (B) Changes of CD45+CD11b+SiglecF+Ly6C in eosinophil proportions are presented using a bar graph. Relative expression levels of (C) IgE, (D) IL-5 and (E) IL-13 in BALF were assessed. *P<0.05, **P<0.01 and ***P<0.001 compared with the control group. BALF, bronchoalveolar lavage fluid.

Fig 5: Flow cytometry analysis of TILs in the TME of syngeneic tumors with wild-type (a) or mutant (b) Trp53. Mice with established MH-22A or MC38 tumors were treated with 10 mg/kg APG-115 (a and b), 10 mg/kg (a) or 5 mg/kg (b) anti-PD-1 antibody, or the combination as described in the legend of Fig. 4. The control group was treated with isotype control antibody and APG-115 vehicle (I + V). On day 14, syngeneic tumors were harvested, dissociated into single-cell suspensions, and stained for flow cytometry analysis. Percentages of CD45+, CD3+ T cells, CD8+ T cells, M1 and M2 macrophages in the tumors under the different treatments were assessed. Data were representative of two (a) or three (b) independent experiments and shown as dot plots (n = 5 or 10). ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05, by one-way ANOVA with Bonferroni post-test. I + V indicates isotype control and vehicle of APG-115