Journal: Cancer Gene Therapy

Article Title: Gut microbiota-mediated activation of GSDMD ignites colorectal tumorigenesis

doi: 10.1038/s41417-024-00796-2

Figure Lengend Snippet: a 5-month-old Cdx2-Cre + / Apc F/+ mice (sporadic CRC model) bearing colorectal tumors were sacrificed, and their tumors were analyzed by Western blotting using an antibody that recognizes both the full-length form (FL) and N’-terminus (N’) of GSDMD. b 2-month-old Cdx2-Cre-ERT2 + / Apc F/F mice (inducible CRC model) were given three daily i.p . injections of 70 mg/kg tamoxifen to ablate both copies of Apc . Mice were sacrificed 1 month after the last tamoxifen injection, and colonic tumors were analyzed by Western blotting. c Human colorectal adenocarcinoma specimens were cryosectioned and stained with antibodies against cleaved GSDMD, Ep-CAM, and DAPI. Images are representative of 4 human CRC cases. d Human cancers of the kidney (KC), lung (LC), bladder (BLC), endometrium (EC), and ovary (OC) were analyzed by Western blotting to detect full-length (FL) and N’-terminus (N’) GSDMD. α-Tubulin blotting was used as a loading control.

Article Snippet: Additional antibodies include IL-1α (Cell Signaling Technology, Cat # 50794 S), IL-1β (Sigma Aldrich, Cat # I3767), cleaved IL-1β (Cell Signaling Technology, Cat # 63124) and IL-18 (BioVision, Cat # 5180R).

Techniques: Western Blot, Injection, Staining, Control

Journal: Cancer Gene Therapy

Article Title: Gut microbiota-mediated activation of GSDMD ignites colorectal tumorigenesis

doi: 10.1038/s41417-024-00796-2

Figure Lengend Snippet: a 5-month-old Cdx2-Cre + / Apc F/+ mice that harbor heterozygous ( Gsdmd +/− , used as controls) or null ( Gsdmd −/− ) alleles of GSDMD were sacrificed and subjected to colorectal tumor statistics. Tumor load was calculated as the summation of tumor diameters for each mouse, n = 19. b Normal colon tissue and colorectal tumors from 5-month-old Cdx2-Cre + / Apc F/+ mice harboring Gsdmd +/− or Gsdmd −/− alleles were dissected and analyzed by Western blotting. c , d Bone marrow cells were harvested from Gsdmd +/+ and Gsdmd −/− mice, and transferred into lethally irradiated 6–8-week-old Cdx2-Cre + / Apc F/+ ( n = 31) mice ( c ) or Cdx2-Cre-ERT2 + / Apc F/F ( n = 22) mice ( d ). c Cdx2-Cre + / Apc F/+ mice were sacrificed at 5 months of age for tumor statistics. d Cdx2-Cre-ERT2 + / Apc F/F mice received 3 doses of 75 mg/kg tamoxifen 2 months following bone marrow transfer and were sacrificed 1 month later. The numbers of visible colonic tumors were counted and shown. e 6–8-week-old Cdx2-Cre + / Apc F/+ mice received bone marrow cell transfer from Gsdmd +/+ or Gsdmd −/− donors, and were sacrificed at 5 months of age. Their colorectal tumors were dissected and analyzed by Western blotting. Each lane represents the pooled tumor lysate of one tumor-bearing mouse. * p < 0.05.

Article Snippet: Additional antibodies include IL-1α (Cell Signaling Technology, Cat # 50794 S), IL-1β (Sigma Aldrich, Cat # I3767), cleaved IL-1β (Cell Signaling Technology, Cat # 63124) and IL-18 (BioVision, Cat # 5180R).

Techniques: Western Blot, Irradiation

Journal: Cancer Gene Therapy

Article Title: Gut microbiota-mediated activation of GSDMD ignites colorectal tumorigenesis

doi: 10.1038/s41417-024-00796-2

Figure Lengend Snippet: a , b , d 5-month-old Cdx2-Cre + / Apc F/+ mice that harbor heterozygous ( Gsdmd +/− ) or null ( Gsdmd −/− ) alleles of GSDMD were sacrificed. Their mesenteric lymph nodes (MLN) and colorectal tumors were digested with collagenase, stained with fluorescent-conjugated antibodies and a live/dead dye, and analyzed by flow cytometry ( n = 7). a Representative flow cytometry staining of Gr-1 and Ly-6C staining of dissociated colorectal tumor cells gated on live cells/CD45 + /CD11b + populations. Right panel shows the statistics of this staining. b Percentages of CD11b + cells in live and CD45 + cells in MLN and colorectal tumors (left panel), and percentages of F4/80 + cells in CD11b + population (right panel). c Tumors were lysed for mRNA extraction and analyzed by bulk RNA sequencing to determine the mRNA levels of arginase 1 (Arg1) and COX2 ( n = 10). d Percentages of CD3 + /CD4 + cells in live and CD45 + cells (left panel), IL-17A + cells in live/CD45 + /CD3 + /CD4 + cells (middle panel), and FOXP3 + cells in live/CD45 + /CD3 + /CD4 + cells in MLN and colorectal tumors. e , f Bone marrow cells were harvested from WT and Gsdmd −/− mice and transferred into lethally irradiated 6–8-week-old Cdx2-Cre + / Apc F/+ mice ( n = 14, e ) or Cdx2-Cre-ERT2 + / Apc F/F ( n = 8, f ) mice. e Bone marrow recipient mice were sacrificed at 5 months of age, and their MLN and tumors were analyzed by flow cytometry. f Bone marrow recipient mice further received tamoxifen injection two months following bone marrow transfer and were sacrificed one month later. MLN and tumor tissues were harvested from these mice and analyzed by flow cytometry. * p < 0.05.

Article Snippet: Additional antibodies include IL-1α (Cell Signaling Technology, Cat # 50794 S), IL-1β (Sigma Aldrich, Cat # I3767), cleaved IL-1β (Cell Signaling Technology, Cat # 63124) and IL-18 (BioVision, Cat # 5180R).

Techniques: Staining, Flow Cytometry, Extraction, RNA Sequencing, Irradiation, Injection

Journal: Cancer Gene Therapy

Article Title: Gut microbiota-mediated activation of GSDMD ignites colorectal tumorigenesis

doi: 10.1038/s41417-024-00796-2

Figure Lengend Snippet: a 5-month-old Cdx2-Cre + Apc F/+ mice were sacrificed, and their colorectal tumors were cryosectioned, followed by immunostaining of indicated antibodies and analysis by confocal microscopy. Representative pictures of 5 mice were shown. Scale bars = 20 μm. b – g 5-month-old Cdx2-Cre + / Apc F/+ mice bearing colorectal tumors were given i.p . injection of NLRP3 inhibitor MCC950 (10 mg/kg) once every 2 days for 7 days. Mice were sacrificed 1 day after the last dose of MCC950 injection, and their mesenteric lymph nodes (MLN), spleen, and colorectal tumors were harvested for analyses. c Tumors from the mice injected with MCC950 or PBS were dissected and analyzed by Western blotting. Each lane represents the pooled tumor lysate of one tumor-bearing mouse. d Quantified levels of cleaved caspase 1 and N’-GSDMD in tumors. PBS: n = 8, MCC950: n = 11. e – g Cells from mouse MLN, spleen, and tumors were dissociated, stained with fluorescent-conjugated antibodies and a live/dead dye, and analyzed by flow cytometry ( n = 6). * p < 0.05 and ** p < 0.01.

Article Snippet: Additional antibodies include IL-1α (Cell Signaling Technology, Cat # 50794 S), IL-1β (Sigma Aldrich, Cat # I3767), cleaved IL-1β (Cell Signaling Technology, Cat # 63124) and IL-18 (BioVision, Cat # 5180R).

Techniques: Immunostaining, Confocal Microscopy, Injection, Western Blot, Staining, Flow Cytometry

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