cdx2 cre ert2 mice  (Jackson Laboratory)

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: C57BL/6, Apc F/F , Cdx2-Cre , and Cdx2-Cre-ERT2 mice were obtained from the Jackson Laboratory.

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: C57BL/6, Apc F/F , Cdx2-Cre , and Cdx2-Cre-ERT2 mice were obtained from the Jackson Laboratory.

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: C57BL/6, Apc F/F , Cdx2-Cre , and Cdx2-Cre-ERT2 mice were obtained from the Jackson Laboratory.

Techniques: Staining, Flow Cytometry, Extraction, RNA Sequencing Assay, 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: C57BL/6, Apc F/F , Cdx2-Cre , and Cdx2-Cre-ERT2 mice were obtained from the Jackson Laboratory.

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

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: 2-month-old Cdx2-Cre-ERT2 + / Apc F/F mice were given i.p . injection of tamoxifen (75 mg/kg) daily for 3 consecutive days. 2 weeks after the last dose of tamoxifen, mice were treated with a cocktail of antibiotics in drinking water (100 μg/ml neomycin, 50 μg/ml vancomycin, 50 μg/ml imipenem, 100 μg/ml metronidazole, 50 μg/ml streptomycin and 100 units/ml penicillin) for three weeks. Fresh antibiotics were given every week. a Scheme of tamoxifen and antibiotic treatment. b Representative Western blotting of colorectal tumors from mice treated by normal drinking water or antibiotics. c Quantified levels of cleaved (N’-terminal) GSDMD in tumors. n = 7 for water, and n = 9 for antibiotics treated mice. d Mice were sacrificed at the end of 3-week antibiotic treatment, and their colorectal tumors were measured by a caliper. Total number of tumors (left panel) and the number of tumors with a diameter equal or larger than 2 mm (right panel) are shown ( n = 12). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: C57BL/6, Apc F/F , Cdx2-Cre , and Cdx2-Cre-ERT2 mice were obtained from the Jackson Laboratory.

Techniques: Injection, Western Blot

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 – c 5-month-old Cdx2-Cre + / Apc F/+ mice that harbor heterozygous ( Gsdmd +/− ) or null ( Gsdmd −/− ) alleles of GSDMD were sacrificed. Their colorectal tumors were cryosectioned and stained for indicated markers. a Representative staining of Gsdmd +/− / Cdx2-Cre + / Apc F/+ tumors. b Human colorectal adenocarcinoma specimens were cryosectioned and stained with indicated markers. Representative images from 4 specimens are shown. c Numbers of CHMP2A ( n = 6) and CHMP4B ( n = 9) aggregates per field were analyzed by immunostaining of these markers on cryosectioned mouse tumors. Scale bars = 10 μm. * p < 0.05 and ** p < 0.01.

Article Snippet: C57BL/6, Apc F/F , Cdx2-Cre , and Cdx2-Cre-ERT2 mice were obtained from the Jackson Laboratory.

Techniques: Staining, Immunostaining

Journal: Research Square

Article Title: An enteroendocrine-microbial axis in the large intestine controls host metabolism

doi: 10.21203/rs.3.rs-3112286/v1

Figure Lengend Snippet:

Article Snippet: These Neurog3 fl/fl mice were mated with animals containing the CDX2 Cre-ERT2 transgene and available from Jackson (JAX Strain #035169) .

Techniques: SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Software