Fig 1: DOK3 regulates S100a8/9 production by neutrophils.A Expression heatmap based on microarray analysis of genes differentially expressed between colonic cells from WT and Dok3-/- mice given 2% DSS in drinking water for 6 days. B Immunoblot analysis of S100a8, S100a9, and GAPDH in untreated (-) or cecal content (CC)-treated (+) lamina propria cells from WT and Dok3-/- mice. Data is representative of three independent experiments. C RT-qPCR analysis of S100a8 and S100a9 expression relative to b-actin expression in lamina propria cells of WT and Dok3-/- mice following 3 h stimulation with cecal contents from WT and Dok3-/- mice. Data is shown as mean±S.E.M (n = 4, four independent experiments). ***p < 0.0001, two-way ANOVA. D RT-qPCR analysis of S100a8 and S100a9 expression relative to b-actin expression in CD45- and CD45+ cell fractions isolated from the lamina propria of WT and Dok3-/- mice following 3 h stimulation with cecal contents from WT mice. Data is shown as mean ± SEM (n = 5, five independent experiments). *p = 0.04, **p = 0.002, unpaired two-tailed Student’s t-test. E Flow cytometric analysis of S100a8 expression in WT and Dok3-/- neutrophils following 3 h stimulation with cecal contents from WT mice. Histograms were pre-gated on singlet, CD45+, Ly6G+ cells. Filled histogram represent isotype control. Bar graph depicting MFI of S100a8 fluorescence in Dok3-/- neutrophils relative to WT neutrophils. Data is shown as mean ± SD (n = 6, five independent experiments). **p = 0.004, unpaired two-tailed Student’s t-test. F Flow cytometric analysis of S100a8 expression in co-housed WT and Dok3-/- neutrophils following 3 h stimulation with cecal contents from WT mice. Histograms were pre-gated on singlet, CD45+, Ly6G+ cells. Filled histogram represent isotype control. Bar graph depicting MFI of S100a8 fluorescence in Dok3-/- neutrophils relative to WT neutrophils. Data is shown as mean ± SD (n = 4, three independent experiments). **p < 0.0001, unpaired two-tailed Student’s t-test. G RT-qPCR analysis of S100a8 and S100a9 expression relative to b-actin expression in bone marrow-purified neutrophils from WT and Dok3-/- mice following 3 h stimulation with cecal contents from WT mice. Data is shown as mean±S.E.M (n = 6, six independent experiments). *p = 0.02, **p = 0.003, unpaired two-tailed Student’s t-test. H WT and Dok3-/- mice were injected intraperitoneally with RAGE antagonist (1 mg/kg) daily, and given 2% DSS in their drinking water for 7 days. Body weights were measured daily. Data is shown as mean ± SEM (n = 4). ***p < 0.0001, two-way repeated-measures ANOVA. I WT and Dok3-/- mice were injected intraperitoneally with Paquinimod (5 mg/kg) daily, and given 2% DSS in their drinking water for 8 days. Body weights were measured daily. Data is shown as mean ± SEM (n = 4). *p = 0.03, ***p < 0.0001, two-way repeated-measures ANOVA.