Fig. 3.

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