Journal: PloS one

Article Title: Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts.

doi: 10.1371/journal.pone.0010622

Figure Lengend Snippet: Figure 1. Brg1 is required for repression of Oct4 expression at the blastocyst stage. (A) qRT-PCR analysis of Brg1, Cdx2, and Oct4 transcripts in Brg1 KD 8-cell embryos, morulae, and blastocysts. Data were normalized to Ubtf (house keeping gene) and are relative to control embryos at each stage; black line = 1. Asterisk denotes significant difference between Brg1 KD and control blastocysts (p,0.05). (B) ICC analysis of Oct4 and Cdx2 expression in Brg1 KD 8-cell embryos, morulae, and blastocysts. Nuclei were counter stained with DAPI (blue). (C) Brg1 represses Oct4 expression in a dose dependent manner. One-cell embryos were injected with 0 mM (control), 0.1 mM, 1 mM, or 100 mM Brg1 siRNA and cultured to the blastocyst stage. Real-time qPCR was used to analyze the levels of Brg1 and Oct4 transcripts. Data were normalized to Ubtf and are relative to control blastocysts; dashed line = 1. doi:10.1371/journal.pone.0010622.g001

Article Snippet: CDX2 was detected by Western blot analysis using an affinity purified rabbit anti-CDX2 antibody (A300-692A, Bethyl Laboratories, Inc).

Techniques: Expressing, Quantitative RT-PCR, Control, Staining, Injection, Cell Culture

Journal: PloS one

Article Title: Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts.

doi: 10.1371/journal.pone.0010622

Figure Lengend Snippet: Figure 2. Expression and localization of Cdx2 and Oct4 in Brg1 KD blastocysts. (A) ICC analysis of Oct4 and Cdx2 in Brg1 KD and control blastocysts. In control blastocysts (a–d) Oct4 expression (green) is restricted to the ICM and is largely absent in the Cdx2-positive (red) trophectoderm. In contrast, in Brg1 KD blastocysts (e–h) Oct4 is widely expressed in both the ICM and cdx2-positive (yellow) trophectoderm. Arrowheads denote co- expression of Oct4 and Cdx2. Nuclei were counter stained with DAPI (blue). (B) Quantification of the average number of cells in Brg1 KD and control blastocysts expressing Oct4, Cdx2, and Oct4 & Cdx2 (double expression). Asterisks denote statistical significance (p,0.05) between Brg1 KD and control blastocysts. A total of 25 Brg1 KD blastocysts and 15 control blastocysts were analyzed. doi:10.1371/journal.pone.0010622.g002

Article Snippet: CDX2 was detected by Western blot analysis using an affinity purified rabbit anti-CDX2 antibody (A300-692A, Bethyl Laboratories, Inc).

Techniques: Expressing, Control, Staining

Journal: PloS one

Article Title: Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts.

doi: 10.1371/journal.pone.0010622

Figure Lengend Snippet: Figure 5. Model for Brg1/Cdx2-mediated repression of Oct4 expression in trophectoderm. Schematic diagram of Oct4 regulation in blastocysts. In the trophectoderm Brg1 is recruited to the ARE of the Oct4 promoter via Cdx2. Once at the Oct4 promoter Brg1 and Cdx2 facilitate recruitment of additional co-repressors to repress transcription. doi:10.1371/journal.pone.0010622.g005

Article Snippet: CDX2 was detected by Western blot analysis using an affinity purified rabbit anti-CDX2 antibody (A300-692A, Bethyl Laboratories, Inc).

Techniques: Expressing

Journal: The International journal of developmental biology

Article Title: Chromatin organization and timing of polar body I extrusion identify developmentally competent mouse oocytes.

doi: 10.1387/ijdb.180362sg

Figure Lengend Snippet: Fig. 4. Immuno-detection of CDX2- and OCT4-positive blastomeres in blastocysts. Blastocysts developed from fertilized group A or ovulated MII oocytes. Blastocysts were counterstained with DAPI used to count the total number of blastomeres. The number of blastomeres was not significantly different (p> 0.05) among the two groups analyzed. Bar, 10 mm.

Article Snippet: Embryos were then processed for sequential immunolabeling using a rabbit polyclonal anti-human OCT4 (Abcam, cat. n. ab19857) and a rabbit anti-human CDX2 (Cell Signaling, cat. n 3977S) antibody.

Techniques:

Journal: Molecular reproduction and development

Article Title: Loss of KANSL3 leads to defective inner cell mass and early embryonic lethality

doi: 10.1002/mrd.23760

Figure Lengend Snippet: Assisted hatching partially rescues the mutant phenotype. (a) Outgrowth assays performed on blastocysts from heterozygous intercrosses after zona pellucida removal. Representative images of wildtype (WT), Heterozygous (HET), and mutant (MUT) embryos and their corresponding outgrowths are shown. Three mutant embryos are shown to reveal consistently abnormal ICM morphology. Scale bars, 100 μm. (b) Immunofluorescence against OCT4 (ICM) in green and CDX2 (TE) in red with DAPI as nuclear stain in blue on embryo outgrowths. Representative IF images of control (top row) and mutant (bottom row) are shown.

Article Snippet: The primary antibodies used are as follows: Rabbit anti-OCT4 (Abcam Cat# ab200834, RRID:AB_2924374, 1:200); mouse anti-CDX2 (BioGenex Cat# MU392A, RRID:AB_2923402, 1:100); rabbit anti-CDX2 (Abcam Cat# ab76541, RRID:AB_1523334, 1/500); goat-anti SOX2 (R and D Systems Cat# AF2018, RRID:AB_355110, 1:200); rabbit anti-NANOG (ReproCELL Incorporated Cat# RCAB0002P-F, RRID:AB_2616320, 1:500); goat anti-SOX17 (R and D Systems Cat# AF1924, RRID:AB_355060, 1:200); rabbit anti-Histone H3 (phospho S10) (Abcam Cat# ab5176, RRID:AB_304763, 1:200); mouse anti-H4K16ac (Thermo Fisher Scientific Cat# MA5-27794, RRID:AB_2735098, 1:150); Rabbit anti-H4K5ac (Thermo Fisher Scientific Cat# MA5-32009-25UG, RRID:AB_2866507, 1:200), Mouse anti-H4K8ac (Thermo Fisher Scientific Cat# MA5-31553, RRID:AB_2787181, 1:200), Rabbit anti-H4K12 (Active Motif Cat# 39927, RRID:AB_2793396, 1:250).

Techniques: Mutagenesis, Immunofluorescence, Staining, Control

Journal: Molecular reproduction and development

Article Title: Loss of KANSL3 leads to defective inner cell mass and early embryonic lethality

doi: 10.1002/mrd.23760

Figure Lengend Snippet: Kansl3 null mutants display ICM-specific defects. (a) Immunofluorescence of SOX2 (ICM) in red and CDX2 (TE) in white with DAPI as nuclear stain in blue on E3.5 blastocysts. Representative image of control (top row) and mutant (bottom row) embryo. (b) Scatter plot comparing absolute numbers of SOX2 positive cells (13 mutant embryos and 18 control embryos). (c) Scatter plot comparing the percentage of SOX2 positive cells (7 control and 7 mutant embryos). There is a significant reduction in SOX2-positive cells in mutant embryos compared to control littermates. (d) Scatter plot comparing absolute number of CDX2 positive cells (13 mutant and 18 control embryos). (e) Scatter plot comparing the percentage of CDX2 positive cells (7 control and 7 mutant embryos). (f) Scatter plot comparing the total number of cells (DAPI nuclei) in 7 control and 7 mutant embryos. (g) Scatter plot comparing intensity of SOX2 normalized against DAPI intensity in individual nuclei in 8 control and 5 mutant embryos. There is a significant reduction in SOX2 intensity in mutant ICM cells. (h) Representative image of TUNEL staining (green) and nuclei (blue). No obvious TUNEL + cells were observed, regardless of genotype. Statistical significance was calculated by a two-tailed Student’s t-test. Error bars represent standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ICM, inner cell mass. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Article Snippet: The primary antibodies used are as follows: Rabbit anti-OCT4 (Abcam Cat# ab200834, RRID:AB_2924374, 1:200); mouse anti-CDX2 (BioGenex Cat# MU392A, RRID:AB_2923402, 1:100); rabbit anti-CDX2 (Abcam Cat# ab76541, RRID:AB_1523334, 1/500); goat-anti SOX2 (R and D Systems Cat# AF2018, RRID:AB_355110, 1:200); rabbit anti-NANOG (ReproCELL Incorporated Cat# RCAB0002P-F, RRID:AB_2616320, 1:500); goat anti-SOX17 (R and D Systems Cat# AF1924, RRID:AB_355060, 1:200); rabbit anti-Histone H3 (phospho S10) (Abcam Cat# ab5176, RRID:AB_304763, 1:200); mouse anti-H4K16ac (Thermo Fisher Scientific Cat# MA5-27794, RRID:AB_2735098, 1:150); Rabbit anti-H4K5ac (Thermo Fisher Scientific Cat# MA5-32009-25UG, RRID:AB_2866507, 1:200), Mouse anti-H4K8ac (Thermo Fisher Scientific Cat# MA5-31553, RRID:AB_2787181, 1:200), Rabbit anti-H4K12 (Active Motif Cat# 39927, RRID:AB_2793396, 1:250).

Techniques: Immunofluorescence, Staining, Control, Mutagenesis, TUNEL Assay, Two Tailed Test, Standard Deviation

Journal: Molecular reproduction and development

Article Title: Loss of KANSL3 leads to defective inner cell mass and early embryonic lethality

doi: 10.1002/mrd.23760

Figure Lengend Snippet: Kansl3 null mutants have fewer epiblast and primitive endoderm cells. (a) Immunofluorescence of Phospho-H3 (green), SOX2 (ICM, red), and CDX2 (TE white) with DAPI (blue). Representative control and a mutant are shown. (b) Scatter plot comparing the percentage of Phospho-H3 positive cells (5 control and 5 mutant embryos). No significant difference is observed. (c) Immunofluorescence of NANOG (EPI, green), SOX17 (PrE) in white and CDX2 (TE, red) with DAPI as nuclear stain. Representative control mutant shown. (d) Scatter plot comparing the percentage of SOX17 positive cells (6 control and 6 mutant embryos). There is a significant reduction in SOX17 cells in mutant embryos. (e) Scatter plot comparing the percentage of NANOG-positive cells (6 control and 6 mutant embryos). There is a significant reduction in NANOG-positive cells in mutant embryos. Statistical significance was calculated by a two-tailed Student’s t-test. Error bars represent standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ICM, inner cell mass.

Article Snippet: The primary antibodies used are as follows: Rabbit anti-OCT4 (Abcam Cat# ab200834, RRID:AB_2924374, 1:200); mouse anti-CDX2 (BioGenex Cat# MU392A, RRID:AB_2923402, 1:100); rabbit anti-CDX2 (Abcam Cat# ab76541, RRID:AB_1523334, 1/500); goat-anti SOX2 (R and D Systems Cat# AF2018, RRID:AB_355110, 1:200); rabbit anti-NANOG (ReproCELL Incorporated Cat# RCAB0002P-F, RRID:AB_2616320, 1:500); goat anti-SOX17 (R and D Systems Cat# AF1924, RRID:AB_355060, 1:200); rabbit anti-Histone H3 (phospho S10) (Abcam Cat# ab5176, RRID:AB_304763, 1:200); mouse anti-H4K16ac (Thermo Fisher Scientific Cat# MA5-27794, RRID:AB_2735098, 1:150); Rabbit anti-H4K5ac (Thermo Fisher Scientific Cat# MA5-32009-25UG, RRID:AB_2866507, 1:200), Mouse anti-H4K8ac (Thermo Fisher Scientific Cat# MA5-31553, RRID:AB_2787181, 1:200), Rabbit anti-H4K12 (Active Motif Cat# 39927, RRID:AB_2793396, 1:250).

Techniques: Immunofluorescence, Control, Mutagenesis, Staining, Two Tailed Test, Standard Deviation

Journal: Molecular reproduction and development

Article Title: Loss of KANSL3 leads to defective inner cell mass and early embryonic lethality

doi: 10.1002/mrd.23760

Figure Lengend Snippet: Kansl3 mutants display lower levels of H4k5ac. (a) Immunofluorescence of H4k5ac (green), SOX2 (ICM, red), and CDX2 (white) with DAPI as a nuclear stain (blue). Representative control (top row) and mutant (bottom row) embryo. (b) Scatter plot comparing the intensity of H4k5ac normalized against DAPI in individual nuclei (3 control and 4 mutant embryos). (c) Immunofluorescence of H4k8ac (white), SOX2 (ICM, red), and CDX2 (green) with DAPI as a nuclear stain (blue). Representative control (top row) and mutant (bottom row) embryo. (d) Scatter plot comparing the intensity of H4k8ac normalized against DAPI in individual nuclei (3 control and 3 mutant embryos). (e) Immunofluorescence of H4k12ac (green) and CDX2 (white) with DAPI as nuclear stain (blue). Representative control (top row) and mutant (bottom row) embryo. (f) Scatter plot comparing the intensity of H4k12ac normalized against DAPI in individual nuclei (3 control and 3 mutant embryos). (g) Immunofluorescence of SOX2 (ICM, red) and H4k16ac (white) with DAPI as a nuclear stain (blue). Representative control (top row) and mutant (bottom row) embryo. (h) Scatter plot comparing the intensity of H4k16ac normalized against DAPI in individual nuclei (4 control and 2 mutant embryos). No significant difference is observed. Statistical significance was calculated by a two-tailed Student’s t-test. Error bars represent standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The primary antibodies used are as follows: Rabbit anti-OCT4 (Abcam Cat# ab200834, RRID:AB_2924374, 1:200); mouse anti-CDX2 (BioGenex Cat# MU392A, RRID:AB_2923402, 1:100); rabbit anti-CDX2 (Abcam Cat# ab76541, RRID:AB_1523334, 1/500); goat-anti SOX2 (R and D Systems Cat# AF2018, RRID:AB_355110, 1:200); rabbit anti-NANOG (ReproCELL Incorporated Cat# RCAB0002P-F, RRID:AB_2616320, 1:500); goat anti-SOX17 (R and D Systems Cat# AF1924, RRID:AB_355060, 1:200); rabbit anti-Histone H3 (phospho S10) (Abcam Cat# ab5176, RRID:AB_304763, 1:200); mouse anti-H4K16ac (Thermo Fisher Scientific Cat# MA5-27794, RRID:AB_2735098, 1:150); Rabbit anti-H4K5ac (Thermo Fisher Scientific Cat# MA5-32009-25UG, RRID:AB_2866507, 1:200), Mouse anti-H4K8ac (Thermo Fisher Scientific Cat# MA5-31553, RRID:AB_2787181, 1:200), Rabbit anti-H4K12 (Active Motif Cat# 39927, RRID:AB_2793396, 1:250).

Techniques: Immunofluorescence, Staining, Control, Mutagenesis, Two Tailed Test, Standard Deviation

Journal: Advanced Science

Article Title: Prediction of Clinical Precision Chemotherapy by Patient‐Derived 3D Bioprinting Models of Colorectal Cancer and Its Liver Metastases

doi: 10.1002/advs.202304460

Figure Lengend Snippet: Study process and histopathological characterization of CRC 3DP models. A) The study process in primary colorectal cancer. A total of 40 patients undergoing surgery for CRC were enrolled, and 3DP models were successfully established and stably cultured for 37 patients. B) Bright‐field images of CRC 3DP models and CRC PDTOs on day 6 after production. Scale bar = 100 µm. C) HE staining comparing CRC 3DP models with corresponding organoids and parental tumors. Scale bar of tumor, 100 µm. Organoid and 3DP scale bars, 20 µm. D) CRC 3DP models and corresponding organoids and parent tumors were co‐stained with CK20 (green), CK7 (red), CDX2 (green), CK‐pan (red), β‐catenin (red), Ki‐67 (green) to examine the profiles of CRC biomarkers. DAPI was used to visualize nuclei (blue). Scale bar of tumor staining = 50 µm. Scale bars of 3DP and organoids staining = 20 µm.

Article Snippet: Rabbit anti‐CK20 (1:200, Abcam, Cambridge, MA, USA), mouse anti‐CK7 (1:200, Abcam), rabbit anti‐CDX2 (1:200, Abcam), rabbit anti‐β‐catenin (1:200, Abcam), mouse anti‐Ki‐67 (1:200, Abcam), and mouse anti‐CK‐pan (1:200, Abcam) were used as primary antibodies for immunohistochemical staining.

Techniques: Stable Transfection, Cell Culture, Staining

Journal: Advanced Science

Article Title: Prediction of Clinical Precision Chemotherapy by Patient‐Derived 3D Bioprinting Models of Colorectal Cancer and Its Liver Metastases

doi: 10.1002/advs.202304460

Figure Lengend Snippet: Histological and genetic mutational characterization of CRLM 3DP models. A) CRLM 3DP models and corresponding organoids and parent tumors were co‐stained with CK20 (green), CK7 (red), CDX2 (green), CK‐pan (red), β‐catenin (red), Ki‐67(green) to examine the profiles of CRC biomarkers. DAPI was used to visualize nuclei (blue). Scale bar of tumor staining = 50 µm. Scale bars of 3DP and organoids staining = 20 µm. B) Proportions of exonic variants for all tested samples (T, tumor; P, 3DP; O, PDTO). C) Spectrum of SNVs in the most frequently mutated genes of CRLM. Each row represents a driver gene, and each column represents the mutational profile of CRLM 3DP models, PDTOs, and parental tumors (T, tumor; P, 3DP; O, PDTO). D) Bar plots present the concordance among the SNVs of the most frequently mutated genes identified in CRLM 3DP models, PDTOs, and corresponding primary tumors.

Article Snippet: Rabbit anti‐CK20 (1:200, Abcam, Cambridge, MA, USA), mouse anti‐CK7 (1:200, Abcam), rabbit anti‐CDX2 (1:200, Abcam), rabbit anti‐β‐catenin (1:200, Abcam), mouse anti‐Ki‐67 (1:200, Abcam), and mouse anti‐CK‐pan (1:200, Abcam) were used as primary antibodies for immunohistochemical staining.

Techniques: Staining