Figure 1.
ERAdP promotes DCV generation in Paneth cells via c-di-AMP stimulation. (A) HE staining of intestines from SPF and GF mice. Scale bar: 20 μm. (B) AB-PAS staining of intestines from SPF and GF mice. Scale bar: 20 μm. (C) HE staining of intestines from control and ABX mice. Scale bar: 20 μm. ABX mice were treated with 2-wk antibiotic, and control mice were treated with water. (D) AB-PAS staining of intestines from control and ABX mice. Scale bar: 20 μm. ABX mice and control mice were treated as in C. (E) Left: Electron micrograph of SPF, ABX, and GF mice. Paneth cells were drawn with dashed white lines, normal DCVs were indicated by yellow asterisk, and abnormal DCVs were indicated by red asterisk. Scale bar: 5 μm. Right: Numbers of DCVs per Paneth cells in SPF, ABX, and GF mice were calculated (n = 10 mice for each group). (F) Left: 3D immunofluorescence staining of ileum crypts from littermate SPF, ABX, and GF mice. Green: UEA-1, red: EpCAM, and yellow: DCVs’ surface fitted by Imaris 9 software. Scale bar: 35 μm. Right: Volume of DCVs was quantified (n = 6 mice for each group). (G) Left: Immunofluorescence staining of intestinal organoids treated with mock, or different stimulations of PRRs: 10 μΜ c-di-AMP disodium, 10 μΜ c-di-GMP disodium, 10 μΜ 3′3′-cGAMP disodium, 10 μΜ 2′3′-cGAMP disodium, 10 μg/ml LPS, 10 μg/ml poly (I:C) sodium, and 2 μg/ml MDP for 16 h. Green: UEA-1, blue: DAPI, and red: EpCAM. Scale bar: 20 μm. Right: Number of DCVs per Paneth cells was quantified (n = 6 mice for each group). (H) Left: Immunofluorescence staining of ileums from mice of control, ABX mock treated, or treated through intragastric gavage with c-di-AMP (25 mg/kg for mouse) twice, with treatments 12 h apart. Green: UEA-1; blue: DAPI, red: EpCAM. Scale bar: 20 μm. Right: Number of DCVs per Paneth cells was quantified (n = 6 mice for each group). (I)Cnep1r1 FISH of 10 different tissues of WT mice. Red: Cnep1r1 probes; blue: DAPI. Scale bar: 35 μm. (J) ERAdP expression levels of indicated tissues from ERAdP-HA tag mice were detected by western blot using anti-HA antibody. β-Actin was used as a loading control. (K)Cnep1r1 FISH of intestine of Lgr5-EGFP mice. Red: Cnep1r1 probes, green: Lgr5EGFP, blue: DAPI, and gray: EpCAM. Scale bar: 35 μm. (L) Immunofluorescence staining showing ERAdP subcellular localization in intestines from ERAdP-HA or WT mice. Red: HA, green: WGA, blue: DAPI, and pink: lysozyme. Scale bar: 15 μm. Data in E–H are shown as means ± SEM, then significance was determined by unpaired two-tailed Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). Data above are representative of at least three independent experiments. Source data are available for this figure: SourceData F1.

ERAdP promotes DCV generation in Paneth cells via c-di-AMP stimulation. (A) HE staining of intestines from SPF and GF mice. Scale bar: 20 μm. (B) AB-PAS staining of intestines from SPF and GF mice. Scale bar: 20 μm. (C) HE staining of intestines from control and ABX mice. Scale bar: 20 μm. ABX mice were treated with 2-wk antibiotic, and control mice were treated with water. (D) AB-PAS staining of intestines from control and ABX mice. Scale bar: 20 μm. ABX mice and control mice were treated as in C. (E) Left: Electron micrograph of SPF, ABX, and GF mice. Paneth cells were drawn with dashed white lines, normal DCVs were indicated by yellow asterisk, and abnormal DCVs were indicated by red asterisk. Scale bar: 5 μm. Right: Numbers of DCVs per Paneth cells in SPF, ABX, and GF mice were calculated (n = 10 mice for each group). (F) Left: 3D immunofluorescence staining of ileum crypts from littermate SPF, ABX, and GF mice. Green: UEA-1, red: EpCAM, and yellow: DCVs’ surface fitted by Imaris 9 software. Scale bar: 35 μm. Right: Volume of DCVs was quantified (n = 6 mice for each group). (G) Left: Immunofluorescence staining of intestinal organoids treated with mock, or different stimulations of PRRs: 10 μΜ c-di-AMP disodium, 10 μΜ c-di-GMP disodium, 10 μΜ 3′3′-cGAMP disodium, 10 μΜ 2′3′-cGAMP disodium, 10 μg/ml LPS, 10 μg/ml poly (I:C) sodium, and 2 μg/ml MDP for 16 h. Green: UEA-1, blue: DAPI, and red: EpCAM. Scale bar: 20 μm. Right: Number of DCVs per Paneth cells was quantified (n = 6 mice for each group). (H) Left: Immunofluorescence staining of ileums from mice of control, ABX mock treated, or treated through intragastric gavage with c-di-AMP (25 mg/kg for mouse) twice, with treatments 12 h apart. Green: UEA-1; blue: DAPI, red: EpCAM. Scale bar: 20 μm. Right: Number of DCVs per Paneth cells was quantified (n = 6 mice for each group). (I)Cnep1r1 FISH of 10 different tissues of WT mice. Red: Cnep1r1 probes; blue: DAPI. Scale bar: 35 μm. (J) ERAdP expression levels of indicated tissues from ERAdP-HA tag mice were detected by western blot using anti-HA antibody. β-Actin was used as a loading control. (K)Cnep1r1 FISH of intestine of Lgr5-EGFP mice. Red: Cnep1r1 probes, green: Lgr5EGFP, blue: DAPI, and gray: EpCAM. Scale bar: 35 μm. (L) Immunofluorescence staining showing ERAdP subcellular localization in intestines from ERAdP-HA or WT mice. Red: HA, green: WGA, blue: DAPI, and pink: lysozyme. Scale bar: 15 μm. Data in E–H are shown as means ± SEM, then significance was determined by unpaired two-tailed Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). Data above are representative of at least three independent experiments. Source data are available for this figure: SourceData F1.

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