Fig 2: Role of ACY1 upregulation in the activation of PTEN/PI3K/AKT signaling. (A) Western blotting assay was used to detect the protein level of p-PI3K, PI3K, p-AKT, AKT, mTOR, and PTEN after 48 hours of cell transfection. (B) RT-PCR analysis of the mRNA level of PI3K, AKT, mTOR and PTEN after 24 hours of cell transfection. (C) IP assay was used to assess the crosstalk between ACY1 protein and PI3K, AKT, mTOR and PTEN proteins (P<0.05). (*, P<0.05 vs. OE-NC group). ACY1, aminoacylase 1; RT-PCR, real-time polymerase chain reaction; PTEN, phosphatase and tensin homolog; IP, immunoprecipitation.

Fig 3: Pik3cg-deficient mice recapitulate major features of human disease after exposure to natural microbiota through co-housing with pet-store mice. a mRNA expression of IL12b normalized to Hprt1 and Rpl13a in murine bone marrow-derived macrophages stimulated with LPS (100 ng/mL) and IFNγ (5 ng/mL) for 24 h. Data from two independent experiments (each with n = 2/group) are presented as mean ± SD. b Supernatant IL-12 concentration from murine bone marrow-derived macrophages stimulated with LPS (100 ng/mL) and IFNγ (5 or 20 ng/mL). Data are presented as mean ± SD and combined from three independent experiments. c Body weight of Pik3cg−/− and wild-type mice co-housed with pet-store mice. Data are representative of four independent experiments. d–f Frequency of Klrg1+ , CD62L+ , and CD44+ among CD8+ T cells from animals with or without exposure to pet-store mice at day 14. Data are presented as mean ± SD and are representative of four independent experiments (n = 3 for each non-co-housed group and n = 8 for each co-housed group). g Frequency of IL-10+ Foxp3+ cells among CD4 + T cells after PMA/ionomycin stimulation of splenocytes from WT (n = 2 for non-co-housed and n = 5 for co-housed) and Pik3cg−/− (n = 2 for non-co-housed and n = 6 for co-housed) after 56 days. Data are presented as mean ± SD. h Concentration of antibodies in serum from animals at 3–5 weeks after exposure to pet-store mice. Data are presented as mean ± SD, summarized from four independent experiments (n = 17–21 for each group). Statistical analysis was performed using unpaired T-test validated with Holm-Sidak method. i Total number of CD3 + T cells by immunohistochemistry per 200 μm section of small intestine from animals with or without exposure to pet-store mice at day 56. Data are presented as mean ± SD and representative of three independent experiments (n = 3 for non-co-housed and n = 7 for co-housed WT; n = 3 for non-co-housed and n = 6 for co-housed Pik3cg−/−). Statistical analysis was performed using two-tailed unpaired T-test in a–g and i

Fig 4: Macrophages/monocytes overproduce inflammatory IL-12 and IL-23 in a GSK3α/β-dependent manner upon TLR stimulation. a mRNA expression of IL12B normalized to GAPDH in monocyte-derived macrophages stimulated with IFNγ (20 ng/mL) and LPS (100 ng/mL) for 24 hr. Data are presented as mean ± SD, and are representative of two independent experiments in which one primary cell isolate for each group was run with three technical replicates. Statistical analysis was performed using a two-tailed unpaired T-test. b–c Immunoblotting for the indicated proteins in lysates from THP-1 monocytes stimulated with LPS (100 ng/mL) and treated with the PI3Kγ inhibitor IPI-549 (1 μM) or DMSO control. Equal protein loading was assessed using stain-free imaging (Bio-Rad). d Schematic of signaling pathway dysregulated in the absence of PI3Kγ activity (created with BioRender.com). e mRNA expression of IL12B normalized to GAPDH in THP-1 macrophages treated with PI3Kγi IPI-549 (1 μM) and GSK3i LY2090314 (20 nM) or DMSO control, as indicated, and stimulated with IFNγ (20 ng/mL) and LPS (100 ng/mL) for 16 h. Data are presented as mean ± SD, and are representative of three independent experiments, in which one primary cell isolate for each group was run with three technical replicates. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test. f mRNA expression of IL12B normalized to GAPDH and RPL37A in THP-1 macrophages expressing PIK3CG-targeted or control shRNA treated as in e and stimulated for 8 hr with IFNγ (20 ng/mL) and LPS (100 ng/mL). Data are presented as mean ± CI, and are representative of two independent experiments in which one primary cell isolate for each group was run with two technical replicates. Statistical analysis was performed using two-tailed unpaired T-test. g–h Cytokine protein concentration (relative to DMSO condition) in supernatant of healthy human donor monocyte-derived macrophages (g) or monocytes (h) treated with IPI-549 (500 nM) and/or GSK3 inhibitor LY2090314 (20 nM), as indicated, and stimulated with LPS (200 ng/mL) and IFNγ (5–35 ng/mL). Data are presented as mean ± SD and combined from 3–4 independent experiments in which one primary cell isolate for each group was run in duplicates. Statistical analysis was performed using two-tailed unpaired T-test