human small intestinal epithelial cell line fhs74int  (ATCC)

Journal: iScience

Article Title: Elevated expression of histone deacetylase HDAC8 suppresses arginine-proline metabolism in necrotizing enterocolitis

doi: 10.1016/j.isci.2023.106882

Figure Lengend Snippet: HDAC8 expression is upregulated in the intestine of humans and mice with NEC (A) Heatmaps of HDAC expression in control and NEC samples (human: control = 8, NEC = 8; mice: control = 3, NEC = 8). (B) HDAC8 protein levels were measured by Western blotting in healthy control and NEC samples. (C) HDAC8 expression was assessed using immunostaining performed on the sections of the terminal ileum in healthy control and NEC samples. Scale bar: 50 μm. (D) qRT‒PCR was performed to assess the relative expression levels of HDAC8 in IECs isolated from humans and mice. ∗p < 0.05, ∗∗p < 0.01 (Mann–Whitney test). (E) qRT‒PCR was performed to assess the relative expression levels of HDAC8 in FHs74Int and IEC6 cells. ∗∗p < 0.01, and ∗∗∗p < 0.001 (two-tailed Student’s t test). (F) qRT‒PCR was performed to assess the relative expression levels of HDAC8 in LPLs isolated from humans and mice. ns means no significance (Mann–Whitney test).

Article Snippet: The non-transformed human fetal intestinal epithelial cell line FHs74Int and the IEC6 cell line were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Control, Western Blot, Immunostaining, Isolation, MANN-WHITNEY, Two Tailed Test

Journal: iScience

Article Title: Elevated expression of histone deacetylase HDAC8 suppresses arginine-proline metabolism in necrotizing enterocolitis

doi: 10.1016/j.isci.2023.106882

Figure Lengend Snippet: HDAC8 regulates the expression of amino acid metabolic enzymes in NEC (A) Analysis of intestinal amino acid concentrations (ug/g) in mice in the control group (n = 3), NEC group (n = 4), PCI34051 group (n = 4), and PCI34051+ NEC group (n = 5). (B and C) qRT‒PCR analysis was performed to assess the relative expression level of enzymes known to be required for arginine synthesis (B) and arginine catabolic process (C) in IECs isolated from intestinal samples obtained from control mice (n = 3), mice with NEC (n = 8), PCI34501-treated mice (n = 6) and PCI34501-treated mice with NEC (n = 7). The results are expressed as the means ± SEMs. Three independent experiments were performed. (D) The levels of enzymes regulated by PCI34051 in the intestine were compared in the indicated groups of FHs74Int cells. The results are expressed as the means ± SEMs. Three independent experiments were performed. (E) Schematic representation of the changes in arginine metabolic enzymes in response to inflammatory conditions (red arrows), PCI34051 treatment (blue arrows), and HDAC8 overexpression (yellow arrows). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns means no significance. Statistical significance was tested by Mann–Whitney test (A, B, C) or two-tailed one-way analysis of variance test (D).

Article Snippet: The non-transformed human fetal intestinal epithelial cell line FHs74Int and the IEC6 cell line were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Control, Isolation, Over Expression, MANN-WHITNEY, Two Tailed Test

Journal: iScience

Article Title: Elevated expression of histone deacetylase HDAC8 suppresses arginine-proline metabolism in necrotizing enterocolitis

doi: 10.1016/j.isci.2023.106882

Figure Lengend Snippet: HDAC8 maintains arginine metabolism enzymes by regulating H3K9ac activity (A) Western blotting analysis was performed to assess the relative expression levels of genes known to be substrates of HDAC8 in healthy controls and NEC samples obtained from humans (upper panel) and mice (lower panel). (B) Western blotting analysis was performed to assess the relative expression levels of genes known to be substrates of HDAC8 in IECs isolated from mice. Three independent experiments were performed. (C) The protein levels of HDAC8, H3K9ac, H3K14ac, H3K27ac, and H4K16ac were analyzed by Western blotting in FHs74Int and IEC6 cells transfected with control lentivirus and HDAC8 lentivirus with 10 μmol/L PCI34051 incubation for 24 h and 10 μg/mL LPS for 48 h. (D) H3K9ac-binding profiles on the targeted gene promoter detected by CUT-Tag in FHs74Int cells. (E) ChIP analysis of H3K9ac in the PRODH and PRODH2 promoters under the conditions of PCI34051 (10 μmol/L for 24 h) or LPS (10 μg/mL for 48 h) treatment in control or HDAC8 stably overexpressing FHs74Int cells. The results are expressed as the means ± SEMs. Three independent experiments were performed. ∗∗p < 0.01, ∗∗∗p < 0.001. ns means no significance. Statistical significance was tested by two-tailed one-way analysis of variance test (E).

Article Snippet: The non-transformed human fetal intestinal epithelial cell line FHs74Int and the IEC6 cell line were obtained from the American Type Culture Collection (ATCC).

Techniques: Activity Assay, Western Blot, Expressing, Isolation, Transfection, Control, Incubation, Binding Assay, Stable Transfection, Two Tailed Test

Journal: iScience

Article Title: Elevated expression of histone deacetylase HDAC8 suppresses arginine-proline metabolism in necrotizing enterocolitis

doi: 10.1016/j.isci.2023.106882

Figure Lengend Snippet: Butyrate inhibits HDAC8 in IECs (A) qRT–PCR analysis of HDAC expression levels in terminal ileum samples obtained from the control mice (n = 3), butyrate-treated mice (n = 7), mice with NEC (n = 8), and butyrate-treated mice with NEC (n = 8). The data are shown in a heatmap. Three independent experiments were performed. (B) Analysis of HDAC8 mRNA levels in IECs isolated from intestinal samples from control mice (n = 3), butyrate-treated mice (n = 7), mice with NEC (n = 8), and butyrate-treated mice with NEC (n = 8). Three independent experiments were performed. (C) FHs74Int and IEC6 cells were incubated with LPS (10 μg/mL, 48 h) or butyrate (2 mmol/L, 12 h) as indicated. HDAC8 mRNA levels were analyzed by qRT–PCR. The results are expressed as the means ± SEMs. Three independent experiments were performed. (D) IHC detection of HDAC8 expression and the expression of molecules known to be substrates of HDAC8 in terminal ileum samples obtained from the different groups as indicated. Scale bar: 50 μm. (E) Western blotting analysis was performed to assess the relative expression levels of HDAC8 and genes known to be substrates of HDAC8 in IECs isolated from the different groups as indicated. (F) The protein levels of HDAC8, H3K9ac, H3K14ac, H3K27ac, and H4K16ac were analyzed by Western blotting in FHs74Int and IEC6 cells transfected with control lentivirus and HDAC8 lentivirus with 10 μg/mL LPS for 48 h and 2 mmol/L butyrate stimulation for 12 has indicated. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns means no significance. Statistical significance was tested by two-tailed one-way analysis of variance test (A, B) or Mann–Whitney test (C).

Article Snippet: The non-transformed human fetal intestinal epithelial cell line FHs74Int and the IEC6 cell line were obtained from the American Type Culture Collection (ATCC).

Techniques: Quantitative RT-PCR, Expressing, Control, Isolation, Incubation, Western Blot, Transfection, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Elevated expression of histone deacetylase HDAC8 suppresses arginine-proline metabolism in necrotizing enterocolitis

doi: 10.1016/j.isci.2023.106882

Figure Lengend Snippet:

Article Snippet: The non-transformed human fetal intestinal epithelial cell line FHs74Int and the IEC6 cell line were obtained from the American Type Culture Collection (ATCC).

Techniques: Sonication, Chromatin Immunoprecipitation, SYBR Green Assay, Software