Figure S1.
c-di-AMP stimulation invivo and expression of Cnep1r1. (A) Quantification of DCV numbers per Paneth cell of SPF and GF mice (n = 6 mice for each group). (B) Intestinal lumen bacterial load analysis, quantified by qPCR of 16S rRNA gene copy number in distal ileums of control (Ctrl) and ABX mice for assessment of commensal bacteria clearance (n = 6 mice for each group). (C) Quantification of DCV numbers per Paneth cell of Ctrl and ABX mice (n = 6 mice for each group). (D) Small intestinal concentration of c-di-AMP, c-di-GMP, and cGAMP from Ctrl, ABX mock, and ABX with these ligands treatment mice was measured by ELISA (n = 6 mice for each group). (E) Immunofluorescence staining of ileums from ABX mice treated through intragastric gavage with c-di-AMP disodium, c-di-GMP disodium, 3′3′-cGAMP disodium, 2′3′-cGAMP disodium, LPS, poly (I:C) sodium, and MDP (25 mg/kg for mouse mouse) twice, with treatments 12 h apart. Green: UEA-1, blue: DAPI, and red: EpCAM. Scale bar: 20 μm. (F) Conservation analysis of ERAdP in indicated vertebrate animals. (G) Relative Cnep1r1 expression levels of indicated tissues were detected by qRT-PCR. Fold changes were normalized to endogenous 18S (n = 3 mice for each group). (H) Construction diagram of ERAdP-HA tag mouse. (I) DNA electrophoresis for ERAdP-HA tag knock-in validation. (J) DNA sequencing for ERAdP-HA tag knock-in validation. (K) Western blot for ERAdP-HA tag knock-in validation. β-Actin was used as a loading control. (L) Immunofluorescence staining of ileal crypts from mice of control, ABX mock treated, or treated through intragastric gavage with c-di-AMP (25 mg/kg for mouse mouse) twice, with treatments 12 h apart. Red: ERAdP-HA, green: UEA-1, blue: DAPI, and gray: EpCAM. Scale bar: 5 μm. (M) Immunofluorescence staining of ileal crypts from WT mice. Green: ERAdP-HA, blue: WGA, and red: PDI. Scale bar: 5 μm. Data in A–D are shown as means ± SEM; significance was determined by unpaired two-tailed Student’s t test (*P < 0.05; ***P < 0.001; ns, not significant). Data in A–E, G, and I–M are representative of at least three independent experiments. Source data are available for this figure: SourceData FS1.

c-di-AMP stimulation invivo and expression of Cnep1r1. (A) Quantification of DCV numbers per Paneth cell of SPF and GF mice (n = 6 mice for each group). (B) Intestinal lumen bacterial load analysis, quantified by qPCR of 16S rRNA gene copy number in distal ileums of control (Ctrl) and ABX mice for assessment of commensal bacteria clearance (n = 6 mice for each group). (C) Quantification of DCV numbers per Paneth cell of Ctrl and ABX mice (n = 6 mice for each group). (D) Small intestinal concentration of c-di-AMP, c-di-GMP, and cGAMP from Ctrl, ABX mock, and ABX with these ligands treatment mice was measured by ELISA (n = 6 mice for each group). (E) Immunofluorescence staining of ileums from ABX mice treated through intragastric gavage with c-di-AMP disodium, c-di-GMP disodium, 3′3′-cGAMP disodium, 2′3′-cGAMP disodium, LPS, poly (I:C) sodium, and MDP (25 mg/kg for mouse mouse) twice, with treatments 12 h apart. Green: UEA-1, blue: DAPI, and red: EpCAM. Scale bar: 20 μm. (F) Conservation analysis of ERAdP in indicated vertebrate animals. (G) Relative Cnep1r1 expression levels of indicated tissues were detected by qRT-PCR. Fold changes were normalized to endogenous 18S (n = 3 mice for each group). (H) Construction diagram of ERAdP-HA tag mouse. (I) DNA electrophoresis for ERAdP-HA tag knock-in validation. (J) DNA sequencing for ERAdP-HA tag knock-in validation. (K) Western blot for ERAdP-HA tag knock-in validation. β-Actin was used as a loading control. (L) Immunofluorescence staining of ileal crypts from mice of control, ABX mock treated, or treated through intragastric gavage with c-di-AMP (25 mg/kg for mouse mouse) twice, with treatments 12 h apart. Red: ERAdP-HA, green: UEA-1, blue: DAPI, and gray: EpCAM. Scale bar: 5 μm. (M) Immunofluorescence staining of ileal crypts from WT mice. Green: ERAdP-HA, blue: WGA, and red: PDI. Scale bar: 5 μm. Data in A–D are shown as means ± SEM; significance was determined by unpaired two-tailed Student’s t test (*P < 0.05; ***P < 0.001; ns, not significant). Data in A–E, G, and I–M are representative of at least three independent experiments. Source data are available for this figure: SourceData FS1.

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