Journal: Translational Oncology

Article Title: L-theanine's therapeutic effects on colorectal cancer: Mechanistic insights from a 1,2-dimethylhydrazine-induced rat model

doi: 10.1016/j.tranon.2025.102609

Figure Lengend Snippet: L-theanine reversed DMH-induced molecular alterations in colon tissues. A-H: Immunohistochemistry (IHC) was used to evaluate the expression of Calretinin (A and B), MOC-31 (C and D), Villin (E and F), and CDX2 (G and H), in colon tissue sections. L -theanine treatment significantly reversed the DMH-induced alterations. Scale bar, 50 μm. Magnification, 200 × . I-M: Western blot analysis was employed to evaluate the expression levels of EMT-related markers, including snail family transcriptional repressor 1 (SNAIL), vimentin, N-cadherin, and E-cadherin, in colon tissues. L -theanine treatment effectively suppressed the expression of mesenchymal markers (SNAIL, vimentin, and N-cadherin) and restored the expression of epithelial markers (E-cadherin), indicating its inhibitory effect on EMT. β-actin was used as an internal reference protein. ⁎⁎ P < 0.01 vs. Control. # P < 0.05, ## P < 0.01 vs. DMH.

Article Snippet: Subsequently, the sections were treated with the following primary antibodies: Ki67 (Huaan Biotechnology Co., Ltd., Wuhan, China; catalog #HA721115, dilution 1:400), CDX2 (Wuhan Sanying Biotechnology Co., Ltd., Wuhan, China; catalog #82,659–1-RR, dilution 1:400), Calretinin (Wuhan Sanying Biotechnology Co., Ltd., Wuhan, China; catalog #12,278–1-AP, dilution 1:400), MOC-31 (Abcam, Boston, MA, USA; catalog #ab213500, dilution 1:800), Villin (Wuhan Sanying Biotechnology Co., Ltd., Wuhan, China; catalog #66,096–1-Ig, dilution 1:400), G-protein-coupled receptor 41 (GPR41, Wuhan Sanying Biotechnology Co., Ltd., Wuhan, China; catalog #66,811–1-Ig, dilution 1:400), Tumour necrosis factor (TNF)-α (Bosterbio, Wuhan, China; catalog #BA0131, dilution 1:200), interleukin (IL)-1β (Boaosen Biotechnology Co., Ltd, Beijing, China; catalog #bs-0812R, dilution 1:100), IL-6 (Servicebio, Wuhan, China; catalog #GB11117, dilution 1:100), and IL-10 (Boaosen Biotechnology Co., Ltd, Beijing, China; catalog #bs-0698R, dilution 1:100).

Techniques: Immunohistochemistry, Expressing, Western Blot, Control

Journal: Frontiers in Oncology

Article Title: Large-Section Histopathology Can Better Indicate the Immune Microenvironment and Predict the Prognosis of Pancreatic Ductal Adenocarcinoma Than Small-Section Histopathology

doi: 10.3389/fonc.2021.694933

Figure Lengend Snippet: Main reagents and instruments.

Article Snippet: IHC: rabbit anti-CDX2 (monoclonal, EP25) , ZSGB-BIO , ZA-0520.

Techniques: Polymer, Imaging

Journal: Scientific Reports

Article Title: Male mice, caged in the International Space Station for 35 days, sire healthy offspring

doi: 10.1038/s41598-019-50128-w

Figure Lengend Snippet: Characterization of fertilized eggs and F1 mice from AG and MG males. ( a ) Embryogenesis of fertilized eggs. Embryogenesis was observed for 4 days after IVF. ( b ) Immunostaining of OCT4 and CDX2 in blastocysts. OCT4 and CDX2 were used as markers for ICM and TE cells in blastocysts, respectively. Scale bars are 100 µm. (c) Quantification of OCT4- and CDX2-positive cells in blastocysts. Data are the mean ± standard deviation. N.S.: not significant. ( d ) Rates of delivered pups. Fertilized eggs obtained by IVF were transferred into pseudopregnent females. ( e ) Offspring from fertilized eggs with MG spermatozoa. ( f ) Growth curve of F1 mice. Body weight was monitored by postnatal day 63. “M” and “F” show the male and female mice, respectively. ( g ) Litter sizes of F1 × F1 intercrosses. Data are the mean ± standard deviation. N.S.: not significant.

Article Snippet: Specifically, rabbit anti-mouse OCT3/4 polyclonal antibody (a marker for ICM cells, 1:200, MBL Code No. PM048) and mouse anti-CDX2 monoclonal antibody (a maker for TE cells, 1:150, MBL Code No. B-MU392AUC) were used as the primary antibodies.

Techniques: Immunostaining, Standard Deviation

Journal: Oncotarget

Article Title: Chemopreventive activity of GEN-27, a genistein derivative, in colitis-associated cancer is mediated by p65-CDX2-β-catenin axis

doi: 10.18632/oncotarget.7554

Figure Lengend Snippet: A. , B. , D. NF-κB/p65, β-catenin nuclear translocation and protein levels of p-IκB-α, IκB-α, Cyclin D1, PCNA, CDX2, E-cadherin, p-GSK3β (S9), GSK3β, p-β-catenin (S37) and total β-catenin were determined by western blot. Data shown are representative of 3 experiments. C. Effects of the indicated concentrations of GEN-27 and GEN (20μM) on β-catenin transcriptional activity of HCT116 cells. TOP- or FOP-flash luciferase plasmid was co-transfected into HCT116 with renilla luciferase plasmid. Luciferase activity was divided by renilla activity. Results are quoted as relative values vs the control value and plotted as mean ± SD ( n = 3). E. The mRNA expressions of c-Myc, Cyclin D1, PCNA, CDX2, APC and AXIN2 in HCT116 cells from each group were determined by Real-time PCR. Values are expressed as mean ± SD ( n = 5). * P < 0.05, ** P < 0.01, *** P < 0.001 vs . control group at 12h, # P < 0.05, ## P < 0.01, ### P < 0.001 vs . control group at 24h. GEN, genistein; GEN-27, genistein-27.

Article Snippet: The cell lysates were immunoblotted using the following primary antibodies: anti-β-catenin (cat. no. ab32572) (Abcam); anti-NF-κB/P65 (cat. no. 6956S), anti-IκB-α (cat. no. 9242), anti-p-IκB-α (Ser32) (cat. no. 2859) (Cell Signaling Technology); and anti-cyclin D1 (H-295) (cat. no. sc-753) (Santa Cruz Biotechnology); anti-GSK3β (cat. no. BS1402), anti-p-GSK3β (S9) (BS4084), anti-p-β-catenin (S37) (cat. no. BS4739), anti-CDX2 (cat. no. MB0125), anti-PCNA (cat. no. BS6438), anti-Lamin A (cat. no. BS1446), anti-β-actin (D8) (cat. no. AP0731) (Bioworld Technology).

Techniques: Translocation Assay, Western Blot, Activity Assay, Luciferase, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Chemopreventive activity of GEN-27, a genistein derivative, in colitis-associated cancer is mediated by p65-CDX2-β-catenin axis

doi: 10.18632/oncotarget.7554

Figure Lengend Snippet: (Aa, Ba, Ca) Protein levels of p65 (Aa), CDX2 (Ba) and β-catenin (Ca) with or without p65 overexpression A. , CDX2 silencing B. , β-catenin overexpression C. (Ab-c, Bb-c, Cb-c) NF-κB/p65, β-catenin nuclear translocation and protein levels of CDX2, p-β-catenin(S37), total β-catenin, PCNA, and Cyclin D1 were determined by western blot. Data shown are representative of 3 experiments. * P < 0.05, ** P < 0.01,*** P < 0.001. (Ad, Bd, Cc) Real-time qPCR analysis of APC and AXIN2 in HCT116 cells. Values are the mean ± SD ( n = 5). (Ae, Be, Ce) Cell viability of HCT116 human colon cancer cells at the indicated time points from each group was analyzed by MTT assay. Each point represents mean ± SD ( n = 5). * P < 0.05, ** P < 0.01,*** P < 0.001 vs . p65+GEN-27 group (Ad), siCDX2+GEN-27 group (Bd), or β-catenin+GEN-27 group (Cd). GEN, genistein; GEN-27, genistein-27.

Article Snippet: The cell lysates were immunoblotted using the following primary antibodies: anti-β-catenin (cat. no. ab32572) (Abcam); anti-NF-κB/P65 (cat. no. 6956S), anti-IκB-α (cat. no. 9242), anti-p-IκB-α (Ser32) (cat. no. 2859) (Cell Signaling Technology); and anti-cyclin D1 (H-295) (cat. no. sc-753) (Santa Cruz Biotechnology); anti-GSK3β (cat. no. BS1402), anti-p-GSK3β (S9) (BS4084), anti-p-β-catenin (S37) (cat. no. BS4739), anti-CDX2 (cat. no. MB0125), anti-PCNA (cat. no. BS6438), anti-Lamin A (cat. no. BS1446), anti-β-actin (D8) (cat. no. AP0731) (Bioworld Technology).

Techniques: Over Expression, Translocation Assay, Western Blot, MTT Assay

Journal: Oncotarget

Article Title: Chemopreventive activity of GEN-27, a genistein derivative, in colitis-associated cancer is mediated by p65-CDX2-β-catenin axis

doi: 10.18632/oncotarget.7554

Figure Lengend Snippet: A. Effect of TNF-α on cell proliferation of HCT116 and HT29 human colon cancer cells. Cells were treated with various concentrations (1-50 ng/ml) of TNF-α for 24 hours, and cell viability was determined using MTT assay. Values were expressed as mean ± SD ( n = 5). B. HCT116 cells were treated with 25 ng/ml TNF-α for the indicated times. Proteins were collected and NF-κB/p65, β-catenin nuclear translocation and protein levels of p-IκB-α, IκB-α, CDX2, Cyclin D1 and PCNA were determined by western blot. * P < 0.05, ** P < 0.01,*** P < 0.001 vs . control group. C. HCT116 cells were pre-treated with GEN-27 (20μM) and GEN (20 μM) for the indicated hours, and the effect of TNF-α (25 ng/ml, 6h) on β-catenin transcriptional activity was determined using TOP- or FOP-flash TOP/FOP-flash reporter system. Results are quoted as relative values vs the control value and plotted as mean ± SD ( n = 3). D. HCT116 cells were incubated with GEN-27 (20 μM) and GEN (20 μM) for the 24 hours, and then treated with 25ng/ml TNF-α for 6 hours. NF-kB/p65 and β-catenin nuclear translocation were analyzed by immunofluorescence cytochemistry (scale bar, 25 μm). E. , F. NF-κB/p65, β-catenin nuclear translocation and protein levels of p-IκB-α, IκB-α, CyclinD1, PCNA, CDX2, p-β-catenin (S37) and total β-catenin were determined by western blot. * P < 0.05, ** P < 0.01,*** P < 0.001 vs . the TNF-α group in (E); * P < 0.05, ** P < 0.01,*** P < 0.001 in (F). G. , H. The mRNA expressions of CDX2, APC, AXIN2, c-Myc, Cyclin D1 and PCNA in HCT116 cells from each group were determined by Real-time PCR. Values are expressed as mean ± SD ( n = 5). I. - K. Chromatin immunoprecipitation (ChIP) using a p65 antibody (I) or CDX2 antibody (J and K) and negative control IgG antibody in HCT116 cells treated with the indicated factors. Immunoprecipitates were probed with primer pairs located within the CDX2 silencer (I), APC enhancer (J) or AXIN2 (K) enhancer region and analyzed by Real-time PCR. Values are shown as percentage of total input DNA and are represented as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01,*** P < 0.001 vs . the TNF-α group; # P < 0.05, ## P < 0.01, ### P < 0.001. GEN, genistein; GEN-27, genistein-27.

Article Snippet: The cell lysates were immunoblotted using the following primary antibodies: anti-β-catenin (cat. no. ab32572) (Abcam); anti-NF-κB/P65 (cat. no. 6956S), anti-IκB-α (cat. no. 9242), anti-p-IκB-α (Ser32) (cat. no. 2859) (Cell Signaling Technology); and anti-cyclin D1 (H-295) (cat. no. sc-753) (Santa Cruz Biotechnology); anti-GSK3β (cat. no. BS1402), anti-p-GSK3β (S9) (BS4084), anti-p-β-catenin (S37) (cat. no. BS4739), anti-CDX2 (cat. no. MB0125), anti-PCNA (cat. no. BS6438), anti-Lamin A (cat. no. BS1446), anti-β-actin (D8) (cat. no. AP0731) (Bioworld Technology).

Techniques: MTT Assay, Translocation Assay, Western Blot, Activity Assay, Incubation, Immunofluorescence, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control

Journal: Oncotarget

Article Title: Chemopreventive activity of GEN-27, a genistein derivative, in colitis-associated cancer is mediated by p65-CDX2-β-catenin axis

doi: 10.18632/oncotarget.7554

Figure Lengend Snippet: A. The expression of CDX2 was detected by immunohistochemistry. Red arrow, cells with CDX2 loss. Data shown are representative of 3 experiments. B. Expression of β-catenin were analyzed by immunofluorescence cytochemistry. C. NF-κB/p65, β-catenin nuclear translocation and protein levels of p-IκB-α, IκB-α, Cyclin D1, PCNA, CDX2, p-β-catenin (S37) and total β-catenin were determined by western blot. Data shown are representative of 3 experiments. * P < 0.05, ** P < 0.01,*** P < 0.001 vs . AOM+DSS group. D. The mRNA expressions of CDX2, APC, AXIN2, Cyclin D1, c-Myc and PCNA in colon sections were determined by real-time PCR. Values are mean ± SD ( n = 5). E. Schematic diagram depicting the role of GEN-27 on Wnt/β-catenin pathway. GEN-27 inhibits TNF-α-induced phosphorylation of IκB-α, and prevents the nuclear translocation of p65, which increases the protein expression of CDX2. And the up-regulated CDX2 increases APC and AXIN2 gene expression, which activates the destruction complex and promotes the phosphorylation of β-catenin at Ser37. As a consequence, GEN-27 inhibits the target gene expressions including PCNA, Cyclin D1 and c-Myc, prevents the colitis-associated initiation, promotion, and progression of tumor development. * P < 0.05, ** P < 0.01, *** P < 0.001 vs . AOM+DSS group. # P < 0.05, ## P < 0.01. GEN, genistein; GEN-27, genistein-27.

Article Snippet: The cell lysates were immunoblotted using the following primary antibodies: anti-β-catenin (cat. no. ab32572) (Abcam); anti-NF-κB/P65 (cat. no. 6956S), anti-IκB-α (cat. no. 9242), anti-p-IκB-α (Ser32) (cat. no. 2859) (Cell Signaling Technology); and anti-cyclin D1 (H-295) (cat. no. sc-753) (Santa Cruz Biotechnology); anti-GSK3β (cat. no. BS1402), anti-p-GSK3β (S9) (BS4084), anti-p-β-catenin (S37) (cat. no. BS4739), anti-CDX2 (cat. no. MB0125), anti-PCNA (cat. no. BS6438), anti-Lamin A (cat. no. BS1446), anti-β-actin (D8) (cat. no. AP0731) (Bioworld Technology).

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Translocation Assay, Western Blot, Real-time Polymerase Chain Reaction

Journal: Advanced Science

Article Title: Multifocal Organoid Capturing of Colon Cancer Reveals Pervasive Intratumoral Heterogenous Drug Responses

doi: 10.1002/advs.202103360

Figure Lengend Snippet: Antibodies used for immunohistochemistry

Article Snippet: Mouse monoclonal anti‐CDX2 , BioGenex , Cat# AM392; RRID:AB_2650531 , 1:300.

Techniques: