Fig 1: Deletion of Olfm4 exacerbates colitis. (A) The mRNA levels of Il6, Il1β, Mcp1, and Icam1 were measured in HCT116 cells by qRT‒PCR. Data are the means ± SDs. n = 6. *P<0.05, **P<0.01. (B) The mRNA levels of Il6, Il1β, Mcp1, and Icam1 were measured in HCT116 cells by qRT‒PCR. Data are the means ± SDs. n = 6. *P<0.05, **P<0.01, ***P<0.001. (C) The protein levels of OLFM4, p-P65, and p-IKK were measured in HCT116 cells by Western blotting and quantified by ImageJ software. *P<0.05. (D) The protein levels of OLFM4, p-P65, and p-IKK were measured in HCT116 cells by Western blot and quantified by ImageJ software. *P<0.05. (E) Olfm4-/- mice were generated through CRISPR technology. (F) Colon sections from WT mice and Olfm4 KO mice were stained with H&E (left), and inflammation scores (right) were determined to assess injury and inflammation. ***P<0.001, ****P<0.0001. (G) The protein levels of OLFM4, p-P65, and p-IKK and the nuclear level of P65 were measured in the distal colons of WT mice and Olfm4-/- mice by Western blotting and quantified by ImageJ software. *P<0.05. (H) The mRNA levels of Il1β, Il6, Mcp1, and Icam1 were measured in distal colons by qRT‒PCR. Data are the means ± SDs. n = 5-8. *P<0.05. (I) The protein levels of IL-1β, IL-6, MCP1, and ICAM1 were measured in serum by ELISAs. Data are the means ± SDs. n = 5-8. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Fig 2: Inhibiting Notch1 could inhibit the regulatory effect of OLFM4 in colitis. (A) The protein levels of genes related to the inflammatory response and p53-mediated apoptosis detected in HCT116 cells. *P<0.05, **P<0.01. (B) The mRNA expression of Olfm4, bax, Mcp1, and Icam1 was measured by qRT‒PCR. Data are the means ± SDs. n = 6. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (C) Mice were injected with DAPT or DMSO every day during the experimental period. (D) Representative western blot analyses and quantification of the protein levels of genes related to the inflammatory response and p53-mediated apoptosis detected in distal colons. *P<0.05, **P<0.01. (E) Colon sections were stained with H&E (left, scale bar: 50 μm), and inflammation scores (right) were calculated to assess injury and inflammation. TUNEL staining showed apoptotic cells in the mouse colons (scale bar: 50 μm). **P<0.01, ***P<0.001, ****P<0.0001. (F) The mRNA expression of p53, Bcl2, Bax, Il1β, Il6, Mcp1, and Icam1 was detected in the distal colons by qRT‒PCR. Data are the means ± SDs. n = 6. *P<0.05, **P<0.01. (G) The protein levels of IL-1β, IL-6, MCP1, and ICAM1 were measured in serum by ELISAs. Data are the means ± SDs. n = 6. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Fig 3: MMP9 mediates the regulatory effect of OLFM4 on p53. (A) The mRNA expression of Mcp1, Il1ß, and Icam1 was detected in HCT116 cells by qRT?PCR. Data are the means ± SDs. n = 6. *P<0.05, **P<0.01. (B) Representative western blot analyses and quantification of the protein levels of genes related to the inflammatory response and p53-mediated apoptosis in HCT116 cells. *P<0.05. (C) AAV-shMMP9 or AAV-shNC was injected into Olfm4-/- mice or WT littermates one week before DSS treatment. (D) Colon sections were stained with H&E (left, scale bar: 50 µm), and inflammation scores (right) were calculated to assess injury and inflammation. TUNEL staining showed apoptotic cells in the colons of mice (scale bar: 50 µm). *P<0.05, **P<0.01, ****P<0.0001. (E) The mRNA expression of Mmp9, Mcp1, Il1ß, Icam1, Bcl2, and P21 was measured by qRT?PCR. Data are the means ± SDs. n = 6. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (F) The protein levels of IL-1ß, IL-6, MCP1, and ICAM1 were measured in serum by ELISAs. Data are the means ± SDs. n = 5-8. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (G) The protein levels of genes related to the inflammatory response and p53-mediated apoptosis were detected by Western blotting and quantified by ImageJ software. *P<0.05.
Fig 4: OLFM4 regulates UC in a p53-dependent manner. (A) Colon sections were stained with H&E (scale bar: 50 µm) to assess injury and inflammation. TUNEL staining showed apoptotic cells in the mouse colons (scale bar: 50 µm). (B) The mRNA expression of Olfm4, Mmp9, Il1ß, Icam1, and Mcp1 was detected in the distal colons by qRT?PCR. Data are the means ± SDs. n = 6. *P<0.05, **P<0.01. (C) The protein levels of IL-1ß, IL-6, MCP1, and ICAM1 were measured in serum by ELISAs. Data are the means ± SDs. n = 6. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (D) The protein levels of MMP9, p-P65, cl-caspase3, cl-caspase7, and PUMA detected by Western blots in mice. *P<0.05, **P<0.01. (E) Serum FITC-dextran levels of DSS-treated WT, Olfm4-/- mice, and Olfm4 re-expression mice. Data are the means ± SDs. n = 6. *P<0.05, **P<0.01, ****P<0.0001. (F) Colon sections were stained with H&E (scale bar: 50 µm) to assess injury and inflammation. TUNEL staining showed apoptotic cells in the mouse colons (scale bar: 50 µm). (G) The mRNA expression of Il1ß, Icam1, and Mcp1 was detected in the distal colons by qRT?PCR. Data are the means ± SDs. n = 6. *P<0.05, **P<0.01, ***P<0.001. (H) The protein levels of IL-1ß, IL-6, MCP1, and ICAM1 were measured in serum by ELISAs. Data are the means ± SDs. n = 6. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (I) Serum FITC-dextran levels of DSS-treated WT, Olfm4-/- mice, and p53 knockdown Olfm4-/- mice. Data are the means ± SDs. n = 6. *P<0.05, **P<0.01, ****P<0.0001. (J) The mRNA expression of Il1ß, Icam1, and Mcp1 was detected in HCT116 cells by qRT?PCR. Data are the means ± SDs. n = 6. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
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