Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: ANKRD22 Drives Rapid Proliferation of Lgr5 + Cells and Acts as a Promising Therapeutic Target in Gastric Mucosal Injury

doi: 10.1016/j.jcmgh.2021.06.020

Figure Lengend Snippet: ANKRD22 deletion reduces the inflammatory response by inhibiting activation of the macrophages after gastric mucosal injury. ( A and B ) Ankrd22 knockout decreased the number of CD45 + cells in damaged mouse gastric mucosa detected by FCM. Rat IgG2b was used as an isotype control (n = 4). ( C and D ) Effect of Ankrd22 knockout on the expression profiles of inflammatory factors in damaged mouse gastric mucosa detected by Luminex assay (n = 4). ( E ) Ankrd22 knockout reduced IL1α and TNF-α in mouse damaged gastric mucosa detected by ELISA (n = 3). ( A – E ) Ankrd22 +/+ and Ankrd22 -/- mice were examined 24 hours after intragastric administration of EtOH/HCl or saline solution (Ctrl). ( F ) Expression levels of ANKRD22 in the macrophages activated by different concentrations of IFN-γ. THP-1 and RAW264.7 macrophages were stimulated with 0, 50, 80, or 100 ng/mL IFN-γ for 24 hours. Subsequently, the expression of ANKRD22 was determined by qRT-PCR. The data from the 0 ng/mL group were normalized to 1.0. TUBB and Tubb were used as internal references. ( G ) Expression levels of Ankrd22 in the activated mouse macrophages detected by qRT-PCR. Data of the PBS-treated group (Ctrl) were normalized to 1.0. Tubb was used as an internal reference. ( H and I ) Determination of IL1α and TNF-α in supernatant of activated macrophages from Ankrd22 +/+ or Ankrd22 -/- mice by ELISA. ( J and K ) Effects of Ankrd22 knockout on the percentages of CD86 + and CD206 + cells in activated Ankrd22 +/+ and Ankrd22 -/- mouse macrophages detected by FCM. Rat IgG2b was used as an isotype control (n = 3). ( L and M ) Determination of IL12 (p70) and IL10 in the supernatant of activated macrophages from Ankrd22 +/+ or Ankrd22 -/- mice by ELISA. ( E – M ) Macrophages were stimulated with 50 ng/mL IFN-γ or 100 ng/mL LPS or PBS (Ctrl) for 24 hours. The data were analyzed by the Student t test and are presented as means ± SD. ∗ P < .05, ∗ P < .01, and ∗∗∗ P < .001. APC-A, Allophycocyanin Area; FITC-A, Fluorescein Isothiocyanate Area; FSC-A, Forward Scatter Area.

Article Snippet: According to the manufacturer's instructions, the cytokine levels of IL1α, TNF-α, IL12, and IL10 in the samples were determined using ELISA kits (Proteintech).

Techniques: Activation Assay, Knock-Out, Control, Expressing, Luminex, Enzyme-linked Immunosorbent Assay, Saline, Quantitative RT-PCR

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: ANKRD22 Drives Rapid Proliferation of Lgr5 + Cells and Acts as a Promising Therapeutic Target in Gastric Mucosal Injury

doi: 10.1016/j.jcmgh.2021.06.020

Figure Lengend Snippet: ANKRD22 deletion reduces the expression levels of mitochondrial Ca 2+ and cytoplasmic NFAT in macrophages. ( A ) Mitochondrial colocalization of exogenous-expressing ANKRD22 in THP-1 macrophages detected by confocal microscopy. ( B – E ) Ankrd22 knockout reduced the mitochondrial Ca 2+ level in activated mouse macrophages. Confocal microscopy was used to compare the fluorescence intensity and colocalization with mitochondria. Macrophages treated with 50 μmol/L 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) for 24 hours was used as a negative control. The fluorescence intensity was detected by Rhod-2–based FCM. ( F ) Determination of IL1α and TNF-α in supernatant of activated mouse macrophages by ELISA after NFAT inhibition. The activated macrophages were treated with 100 ng/mL NFAT inhibitor for 1 hour. ( B – F ) Macrophages were stimulated with 50 ng/mL IFN-γ or 100 ng/mL LPS or PBS (Ctrl) for 24 hours. ( G ) Determination of TNF-α in the supernatant of activated macrophages by ELISA after NFAT inhibition. The LPS-activated macrophages were treated with 100 ng/mL NFAT inhibitor for 1 hour. ( H ) Detection of NFAT in the activated mouse macrophages by Western blot. ( I ) Detection of NFAT in the activated macrophages by Western blot. ( G – I ) THP-1 macrophages were treated with 100 ng/mL phorbol 12-myristate 13-acetate for 24 hours in advance. ( J ) Effects of Ankrd22 knockout on the expression levels of NFAT in activated mouse macrophages detected by Western blot. Macrophages were stimulated with 50 ng/mL IFN-γ or 100 ng/mL LPS for 24 hours. Macrophages were stimulated with 0, 50, or 100 ng/mL IFN-γ or 0, 0.1, or 1.0 μg/mL LPS for 24 hours. Data are presented as means ± SD and analyzed using the Student t test. ∗ P < .05 and ∗∗ P < .01.

Article Snippet: According to the manufacturer's instructions, the cytokine levels of IL1α, TNF-α, IL12, and IL10 in the samples were determined using ELISA kits (Proteintech).

Techniques: Expressing, Confocal Microscopy, Knock-Out, Fluorescence, Negative Control, Enzyme-linked Immunosorbent Assay, Inhibition, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: ANKRD22 Drives Rapid Proliferation of Lgr5 + Cells and Acts as a Promising Therapeutic Target in Gastric Mucosal Injury

doi: 10.1016/j.jcmgh.2021.06.020

Figure Lengend Snippet: Identification of a lead compound that inhibits the activity of ANKRD22. ( A ) Small molecules interacting with the L122/D132 sites of ANKRD22 predicted by protein ligand interface fingerprint software. ( B ) Expression levels of ANKRD22 in the WT, empty vector–transfected, or ANKRD22/pcDNA3.1(-)–transfected SGC7901 cells detected by Western blot. ( C ) Effect of the E115A/D123A mutant on intracellular Ca 2+ levels in ANKRD22 + gastric cancer SGC7901 cells. The fluorescence intensity was detected using Fluo-4–based FCM. ( D ) Effect of the L122A/D132A mutant on the intracellular Ca 2+ levels in ANKRD22 + gastric cancer SGC7901 cells. The fluorescence intensity was detected by Fluo-4–based FCM. ( E ) Schematic diagram of the chemical structure and acting sites of the ANKRD22 inhibitory lead compound-AV023. ( F ) Effects of AV023 on LGR5 in ANKRD22 + SGC7901 cells detected by qRT-PCR. SGC7901 cells without ANKRD22 overexpression were used as the control groups and the cells were treated with 0, 0.05, 0.2, 0.5, and 1.0 μmol/L AV023 for 24 hours. TUBB was used as an internal reference. ( G ) AV023 increased the clone number of primary mouse gastric epithelial cells in organoid culture. Ankrd22 +/+ mouse gastric EPCs were treated with 0 (Ctrl) or 1.0 μmol/L AV023 for 24 hours. ( H ) AV023 reduced the intracellular Ca 2+ levels in Ankrd22 +/+ mouse gastric epithelial cells. The fluorescence intensity was detected by Fluo-4–based FCM. ( I ) AV023 reduced the intracellular Ca 2+ levels in ANKRD22 + SGC7901 cells. The fluorescence intensity was detected by Fluo-4–based FCM. ( J ) AV023 increased the Wnt pathway activity in Ankrd22 +/+ mouse gastric organoids detected by Western blot. ( K ) AV023 increased the Wnt pathway activity in ANKRD22 + SGC7901 cells detected by Western blot. ( L ) AV023 increased the Wnt transcriptional activity of ANKRD22 + SGC7901 cells detected by TOPflash luciferase reporter assay. ( H – L ) Ankrd22 +/+ and Ankrd22 -/- mouse gastric cells were treated with 0, 0.5, and 1.0 μmol/L AV023 for 24 hours. SGC7901 cells without ANKRD22 overexpression were used as the control groups and the cells were treated with 0, 0.05, 0.2, 0.5, and 1.0 μmol/L AV023 for 24 hours. ( M and N ) Effect of AV023 on the release of IL1α and TNF-α in the activated mouse macrophages detected by ELISA. Mouse macrophages were stimulated with 100 ng/mL LPS for 24 hours, and the control group was not treated with LPS. Cells were treated with 0, 0.05, 0.2, 0.5, and 1.0 μmol/L AV023 for 24 hours. The data were analyzed by the Student t test and are presented as means ± SD. ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001.

Article Snippet: According to the manufacturer's instructions, the cytokine levels of IL1α, TNF-α, IL12, and IL10 in the samples were determined using ELISA kits (Proteintech).

Techniques: Activity Assay, Software, Expressing, Plasmid Preparation, Transfection, Western Blot, Mutagenesis, Fluorescence, Quantitative RT-PCR, Over Expression, Control, Luciferase, Reporter Assay, Enzyme-linked Immunosorbent Assay