Fig 1: Changes in CCR2 in mouse and human monocytes in response to Smo chemical inhibition or gene knockdown.A Flow cytometric contour plots demonstrating the gating strategy for quantification of CD11b+F4/80+Ly6CHiCCR2+ macrophages. B Quantification of flow cytometry analysis for the expression of CD11b+F4/80+Ly6CHiCCR2+ macrophages within tissues and PBMCs collected in response to acetic acid-induced injury. Uninjured controls were subjected to surgery and stomachs exposed to PBS. C Quantitative RT-PCR documenting knockdown of Smoothened (SMOKD) in human-derived monocyte cultures. D Immunofluorescence of M1 or M2 macrophages in control and SMOKD cultures for CCR2 expression (red). E Fluorescence intensity was measured using images captured in (D). Quantitative RT-PCR using RNA collected from (F) control and (G) SMOKD cultures polarized to either M1 or M2 macrophages and treated with vehicle (Veh), MCP-1, Shh, or CX3CL1. *P < 0.05 compared to control or vehicle groups, n = 3 individual biological replicates. Data are shown as the mean ± SEM.
Fig 2: Macrophage migration and CCR2 expression.Migration plots of trajectories in response to A medium and B, C rmShh gradient using macrophages cultured from the bone marrow of control and SmoKO mice. D Macrophage migration is represented as forward migration index (FMI). Representative immunofluorescence stains of actin (green) and nuclei (red) of bone marrow-derived macrophages cultured from E control and F SmoKO mice treated with vehicle, CCL2, or rmShh. Arrows indicate filopodia. G Representative immunofluorescence stain of CCR2 (red) and nuclei (Hoechst, blue) of bone marrow-derived macrophages cultured from control or SmoKO mice and treated with PBS, rmShh, or MCP. H Quantitative RT-PCR of CCR2 expression using RNA collected from control or SmoKO bone marrow-derived mouse macrophages and treated with PBS, Shh, MCP1, or CXC3L1. *P < 0.05 compared to PBS vehicle control, n = 6 individual cultures. Data are shown as the mean ± SEM.
Fig 3: Human gastric organoid/macrophage co-cultures.A Acridine orange assay using gastric organoids treated with either vehicle or histamine. B Shift in F458 (red)/F488 (green) fluorescence ratio was measured in the organoid cultures treated with vehicle or histamine. C Quantitative RT-PCR measuring changes in Shh expression in the gastric organoids treated with vehicle (control) or histamine (Hist). *P < 0.05 compared to control, n = 4 cultures generated from four individual patients. D Bright-field micrographs showing tissue-derived human fundic gastric organoids (huFGOs), PBMC-derived macrophages, and representative co-culture. E Magnetic separation was used to isolate epithelial and immune cells from treated human gastric organoid/macrophage co-cultures. F RT-PCR demonstrating expression of Shh in EpCAM positive and CD68 in macrophage fractions. Data are shown as the mean ± SEM.
Fig 4: Organoid/ILC2/monocyte Interactive Co-Culture Plates.A Interactive Co-Culture Plates were used to culture human gastric-derived organoids and ILC2s in the left chamber and monocytes in the right chamber. Conditioned medium collected from the organoid/ILC2 co-culture was used to measure changes in B Shh, C IL-33, and D IL-13 secretion. E Bright-field micrographs of monocytes after treatment with the vehicle, histamine, histamine + vismodegib, anti-IL-33 immuneutralization antibody + Histamine, or anti-IL-33 alone. F Quantitative RT-PCR was used to measure changes in M1/M2 markers in the monocyte cultures. Expression of M2 markers in monocyte chamber isolated from Veh (1), Vis (2), Hist (3) or Vis+Hist (4), anti-IL33 (5), anti-IL33 + Hist (6), anti-IL13 (7), and anti-IL13 + Hist (8), treated co-cultures. G Immunofluorescence staining for expression of CD44v9 (green), IL-33 (red), and Hoechst (blue) in organoid/ILC2 co-cultures treated with vehicle, histamine or histamine + Vismodegib. H Fluorescence intensity for CD44v9 and IL-33 was quantified in co-cultures. *P < 0.05 compared to vehicle, n = 3 co-cultures prepared from three individual patients. Data are shown as the mean ± SEM.
Fig 5: Expression of Shh and macrophage infiltration in control and LysMCre/SmoKO mice.Immunohistochemistry of Shh expression in A control and B LysMCre/SmoKO mouse stomachs. Shh expression at the ulcer margin of C, D control, and E, F LysMCre/SmoKO mouse stomachs. G Shh concentrations (pg/mL) measured in plasma collected from in control and LysMCre/SmoKO mice. Macrophage numbers (CD11b+F4/80+Ly6Chi) within the uninjured and injured gastric epithelium of H control and I LysMCre/SmoKO mice 1–7 post-ulcer induction. *P < 0.05 compared to day 1 post-injury, n = 6 mice per group. Data are shown as the mean ± SEM.
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