Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Immunostaining of original tumor, low passage, and high passage KCI-MENG1 cells. The original patient-derived tumor ( top row ) showed moderate and patchy immunoreactivity for epithelial membrane antigen (EMA); strong and diffuse immunostaining for progesterone receptor (PR); and a Ki-67 proliferative index of 2–3%. There was also strong immunostaining for N-cadherin and vimentin. KCI-MENG1-LP cells ( middle row ) and KCI-MENG1-HP cells ( bottom row ) maintained expression of EMA, N-cadherin, and vimentin but had significantly reduced PR expression compared to the original tumor. Whereas Ki-67 labeling was found in only a small number of cells in the original tumor and low passage cells, it was positive in virtually all P84 cells. Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Immunostaining, Derivative Assay, Membrane, Expressing, Labeling

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Immunostaining of original patient tumor, low and high passage KCI-MENG1 cells, and subcutaneous xenograft tumor. The original patient-derived tumor showed moderate immunoreactivity for E-cadherin which was maintained in all in vitro and in vivo models. Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Immunostaining, Derivative Assay, In Vitro, In Vivo

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Meningioma cell lines reported in the literature

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Southern Blot, Injection, Expressing, TRAP Assay, Activity Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Translational Medicine

Article Title: Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

doi: 10.1186/s12967-015-0596-8

Figure Lengend Snippet: Human meningioma mouse xenograft model KCI-MENG1-LPSX generated with the spontaneously immortal cell line KCI-MENG1-LP. Tumors from immunocompromised SCID mice were dissected ( a ) and the derivative cell line KCI-MENG1-LPSX CL was generated. The H&E staining of the mouse tumor revealed a pattern of moderately cellular meningothelial cells similar to the original patient tumor ( b ). The KCI-MENG1-LPSX CL cells were composed of the round-shaped cells similar to the high passage parent cell line KCI-MENG1-HP ( c ). The EMA, PR, and N-cadherin IHC of the mouse tumor highly resembled the original patient-derived tumor ( d top row ). The vimentin- and Ki-67-stained cells in the mouse tumor tissue were markedly more abundant and more intensely stained than in the original tumor ( d top row ). KCI-MENG1-LPSX CL cells displayed the same patterns of immunostaining as the high passage parent cell line KCI-MENG1-HP, including the loss of PR staining ( d bottom row ). Scale bar 50 µm.

Article Snippet: Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA).

Techniques: Generated, Staining, Derivative Assay, Immunostaining

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A & B. Primary human fibroblasts were pre-labeled by DiI (red) and MDA-MB-231 (A) or BT549 cells (B) were pre-labeled by DiD (blue). Cells were cocultured for overnight before fixation for IF staining. Cadherin-11 protein was stained by the specific monoclonal primary antibody (clone 16A) followed by the secondary antibody staining (green). Purple arrows are pointing to the cadherin-11 AJs between cancer cells and fibroblasts. Confocal Z-stacks were scanned from the top of the cell to the bottom of the cell. All 2-D images shown were from 3-D Z-stacks maximum projections. C. The schematic to describe the cell invasion assay. D. Fibroblasts and MDA-MB-231 cells were pre-labeled as before. Fibroblasts alone (middle panels), MDA-MB-231 alone (right panels) or Fibroblasts and MDA-MB-231 (left panels) together in a 1:1 ratio were subjected to the cell invasion assay as described in (C). The whole cell population invasion displacements on the X-axis with a direction to the left were labeled between the yellow lines of 0 hr and 16 hr. The green fluorescence from the matrigel was omitted to clearly visualize the cells. Size bar, 100 μm. E, F & G. Cell invasion speed (Distance/time), invasion velocity (Displacement on the X-axis/time) and invasion persistence (Displacement/Distance) were quantitated. *, P < 0.05. H. Time-lapse zoom-in panels from Video 3. Green arrows are pointing at one cancer cell that was invading back-n-forth by attaching to and sliding on the cell bodies of fibroblasts. The red channel imaging offsets were elevated to visualize the long but thin invasive protrusions of fibroblasts. Size bar, 100 μm. I. Cropped and zoom-in panels from (H) to present the details of the invasive protrusions of fibroblasts (yellow arrow heads).

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Labeling, Staining, Invasion Assay, Fluorescence, Imaging

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. Primary human fibroblasts were pre-labeled by DiI (red) and BT549 cells were pre-labeled by DiO (green). BT549 alone spheroid with negative control siRNA or CDH11 siRNA (top panels), and fibroblasts alone spheroid with negative control siRNA or CDH11 siRNA (bottom panels) were subjected to the 3D spheroid cell invasion assay. B. Quantitation for images as in (A). Experiments were repeated 6 times (n=6). *, P < 0.05. C. BT549 and fibroblasts coculture (1:1) spheroid with negative control siRNA or CDH11 siRNA were subjected to the 3D spheroid cell invasion assay. D, E & F. Quantitation for images as in (C). Experiments were repeated 6 times (n=6). *, P < 0.05. Total cell numbers maintained the same in every spheroid. Confocal Z-stacks were scanned from the top of the spheroid to the bottom. All 2-D images shown were from 3-D Z-stacks maximum projections.

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Labeling, Negative Control, Invasion Assay, Quantitation Assay

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. CDH11 (Cadherin-11), ESR1(Estrogen Receptor 1), PGR (Progesterone Receptor) & ERBB2 (Receptor tyrosine-protein kinase erbB-2) expression data in breast cancer cell lines from CCLE (Cancer Cell Line Encyclopedia, The Broad Institute of MIT & Harvard) were plotted into a heat map. B. Kaplan-Meier analysis for breast cancer patients stratified by CDH11 expression for 1075 patients from The Human Protein Atlas database. C-E. Kaplan-Meier plots of overall survival (C, n=626), relapse-free survival (D, n=1764) and distant metastasis free survival (E, n=664) of breast cancer patients in relation to CDH11 expression according to The KM-plotter database. F. Kaplan-Meier plots of distant metastasis free survival (n=68) of ER negative breast cancer patients in relation to CDH11 expression according to The KM-plotter database.

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Expressing

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. Primary human fibroblasts were transduced by GIPZ lentiviral CDH11 shRNA or non-silencing shRNA with a GFP reporter. Stable transductant cells were sorted out by FACS based on the GFP signal. Silencing of CDH11 in these cells was then quantified by RT-qPCR. B. Effect of CDH11 silencing in human fibroblasts on cancer cell growth in the cancer with fibroblast co-implantation xenograft mouse model. 1 × 10 6 of MDA-MB-231-luc cells mixed with 1 × 10 6 of stable non-silencing (blue) or CHD11 silencing (red) primary human fibroblasts were co-implanted into the left fourth mammary fat pad of NOD/SCID mice. The bioluminescence of the MDA-MB-231-Luc cells were measured every 2 weeks. C. Representative in vivo bioluminescence images from (B) at 14 weeks after cancer with fibroblast co-implantation. D. Comparison of tumor volume in mice co-implanted with MDA-MB-231-luc cells and non-silencing or CDH11 silencing primary human fibroblasts. Data were presented as mean ± SD (n=8). P values were determined by two-tailed Student’s t tests (NS, not significant; *, 0.01 < p < 0.05).

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: shRNA, Quantitative RT-PCR, In Vivo, Two Tailed Test

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: A. CDH11 stable overexpression in 4T1 cells (4T1-CDH11) was quantified by RT-qPCR against CDH11 expression levels in 4T1 wildtype cells (4T1-WT). B. 4T1-WT cells or 4T1-CDH11 cells were pre-labeled by DiD (red). NIH3T3 mouse fibroblasts were pre-labeled by DiO (green). 4T1 alone spheroids (top panels), or 4T1 and NIH3T3 coculture (1:1) spheroids (bottom panels) were subjected to the 3D spheroid cell invasion assay as in . Total cell numbers maintained the same in every spheroid. Confocal Z-stacks were scanned from the top of the spheroid to the bottom. All 2-D images shown were from 3-D Z-stacks maximum projections. C. Quantitation for images as in (B). Experiments were repeated 5 times (n=5). NS, not significant; *, P < 0.05. D. Comparison of tumor volume in BALB/c mice implanted with 4T1-WT cells or 4T1-CDH11 cells. 1 × 10 6 of 4T1 cells were implanted into the left fourth mammary fat pad in BALB/c mice. Data were presented as mean ± SD (n=8). E. Comparison of 4T1-WT cell and 4T1-CDH11 cell proliferation in 2D culture in vitro. Same number of cells (46,000 cells) of each group were seeded in one well of a 6-well plate. Cell number was counted at 24 hrs, 48 hrs & 72 hrs (n=3 for each cell group at each time point) after cell seeding. NS, not significant. Note all cells were still not confluent at 72 hrs in each well of a 6-well plate. F. Kaplan-Meier survival curve of BALB/c mice implanted with either 4T1-WT cells or 4T1-CDH11 cells as in (D), n=8. G. micro-MRI imaging of tumor-bearing BALB/c mice from (D) on the 21 st day after implantation of 4T1-WT cells or 4T1-CDH11 cells. Multiple distal metastatic sites in the dorsal neck region lymph nodes (denoted by small arrows) were detected in mice with 4T1-CDH11 tumors. Large arrowheads denote the original tumors at the left fourth mammary fat pad. micro-MRI image Z-stacks were scanned from the dorsal side to the ventral side of the mice. Single plane image section across the dorsal neck region lymph nodes from two representative mice from each group is shown. No distal metastasis was detected in any mice with 4T1-WT tumors in all micro-MRI image Z-stacks.

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Labeling, Invasion Assay, Quantitation Assay, In Vitro, Micro-MRI, Imaging

Journal: bioRxiv

Article Title: Post-EMT: Cadherin-11 mediates cancer hijacking fibroblasts

doi: 10.1101/729491

Figure Lengend Snippet: 1 × 10 6 of 4T1 mouse triple negative breast cancer cells expressing firefly luciferase with or without CDH11 overexpression were implanted into the left fourth mammary fat pad of immunocompetent BALB/c mice (A-C) or NOD/SCID mice (E-G). A. Comparison of whole tumor growth volume between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group in BALB/c mice. B. Comparison of cancer growth (as detected by the firefly luciferase bioluminescence) between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group at 4 weeks after implantation in BALB/c mice. Data were presented as mean ± SD (n=7). C. Representative in vivo bioluminescence images from (B). D. Comparison of 4T1-WT-luc cell and 4T1-CDH11-luc cell proliferation in 2D culture in vitro. Same number of cells (46,000 cells) of each group were seeded in one well of a 6-well plate. Cell number was counted at 24 hrs, 48 hrs & 72 hrs (n=3 for each cell group at each time point) after cell seeding. NS, not significant. Note all cells were still not confluent at 72 hrs in each well of a 6-well plate. E. Comparison of whole tumor growth volume between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group in NOD/SCID mice. F. Comparison of cancer growth (as detected by the firefly luciferase bioluminescence) between the 4T1-WT-luc cells implantation group and the 4T1-CDH11-luc cells implantation group at 4 weeks after implantation in NOD/SCID mice. Data were presented as mean ± SD (n=5). G. Representative in vivo bioluminescence images from (F).

Article Snippet: Mouse cadherin-11 expression plasmide was from Sino biological Inc. (Wayne, PA). pGL4.51[luc2/CMV/Neo] vector encoding the firefly luciferase reporter gene luc2 was from Promega (Madison, WI).

Techniques: Expressing, Luciferase, Over Expression, In Vivo, In Vitro

Journal: Carcinogenesis

Article Title: DUOX2 promotes the progression of colorectal cancer cells by regulating the AKT pathway and interacting with RPL3

doi: 10.1093/carcin/bgaa056

Figure Lengend Snippet: RPL3 reverses the effects of DUOX2 in vitro , and the different genes after DUOX2 knockdown were enriched in the AKT pathway, while RPL3 reversed this change partially. ( A ). Western blotting was performed to detect the DUOX2 protein in DUOX2-overexpressed (pcDNA3.1-DUOX2) and negative control (pcDNA3.1-Control) both in HCT116 and SW480 cells. ( B ) Transwell assays. The migration and invasion ability of the HCT116 and SW480 cells were significantly increased after overexpressing DUOX2, while the overexpression of RPL3 significantly reversed this trend. ( C ) Wound healing assay. The migration rate was derived from the ratio of the difference in wound area at different times to the initial wound area (200×). The tests were performed on SW480 cells. N.S. P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001. ( D ) Differentially expressed genes in DUOX2 knockdown (si1-DUOX2, si2-DUOX2) and negative control (si-NC) by next-generation sequencing as shown in the heatmap. ( E ) Different signaling pathways based on KEGG databases between si1-DUOX2 and si-NC group, or si2-DUOX2 and si-NC group. ( F ) Verification of related genes in PI3K–AKT pathway. ( G ) WB assay. The protein levels of E-cadherin (E-cad), EGFR, AKT, p-AKT, c-MYC after overexpression of DUOX2, or both of DUOX2 and RPL3.

Article Snippet: The antibodies for ubiquitin, GAPDH, E-cadherin and N-cadherin were purchased from Proteintech (Wuhan, China).

Techniques: In Vitro, Knockdown, Western Blot, Negative Control, Control, Migration, Over Expression, Wound Healing Assay, Derivative Assay, Next-Generation Sequencing, Protein-Protein interactions

Journal: Bioengineered

Article Title: Circular RNA circ_0001955 promotes hepatocellular carcinoma tumorigenesis by up-regulating alkaline ceramidase 3 expression through microRNA-655-3p

doi: 10.1080/21655979.2021.2023797

Figure Lengend Snippet: Knockdown of circ_0001955 restrains HCC tumorigenesis in vitro . (a-k) HCCLM3 and Huh-7 cells were transfected with si-circ_0001955 or si-NC. (a) qRT-PCR analysis of circ_0001955 expression in cells. (b-d) Cell proliferation analysis using CCK-8 and colony formation assays. (e, f) Transwell assay for cell migration and invasion. (g) Tube formation assay for cell angiogenesis analysis. (h-j) Flow cytometry for cell cycle distribution and cell apoptosis rate. (j, k) Western blot analysis of the protein levels of E-cadherin, vimentin and N-cadherin. * P < 0.05.

Article Snippet: The primary antibodies included: E-cadherin (ab40772, 1:2000, Abcam), vimentin (ab92547, 1:2000, Abcam), N-cadherin (ab18203, 1:2000, Abcam), ACER3 (1:500, Saierbio, Tianjin, China) and GAPDH (ab181602, 1:1000, Abcam).

Techniques: Knockdown, In Vitro, Transfection, Quantitative RT-PCR, Expressing, CCK-8 Assay, Transwell Assay, Migration, Tube Formation Assay, Flow Cytometry, Western Blot

Journal: Bioengineered

Article Title: Circular RNA circ_0001955 promotes hepatocellular carcinoma tumorigenesis by up-regulating alkaline ceramidase 3 expression through microRNA-655-3p

doi: 10.1080/21655979.2021.2023797

Figure Lengend Snippet: Knockdown of circ_0001955 suppresses HCC tumorigenesis via targeting miR-655-3p. (a-k) HCCLM3 and Huh-7 cells were transfected with si-NC, si-circ_0001955, si-circ_0001955 + anti-miR-NC, or si-circ_0001955 + anti-miR-655-3p. (a-c) Cell proliferation analysis using CCK-8 and colony formation assays. (d, e) Transwell assay for cell migration and invasion. (f) Tube formation assay for cell angiogenesis analysis. (g-i) Flow cytometry assay for cell cycle distribution and cell apoptosis rate. (j, k) Western blot analysis of the protein levels of E-cadherin, vimentin and N-cadherin. * P < 0.05.

Article Snippet: The primary antibodies included: E-cadherin (ab40772, 1:2000, Abcam), vimentin (ab92547, 1:2000, Abcam), N-cadherin (ab18203, 1:2000, Abcam), ACER3 (1:500, Saierbio, Tianjin, China) and GAPDH (ab181602, 1:1000, Abcam).

Techniques: Knockdown, Transfection, CCK-8 Assay, Transwell Assay, Migration, Tube Formation Assay, Flow Cytometry, Western Blot

Journal: Bioengineered

Article Title: Circular RNA circ_0001955 promotes hepatocellular carcinoma tumorigenesis by up-regulating alkaline ceramidase 3 expression through microRNA-655-3p

doi: 10.1080/21655979.2021.2023797

Figure Lengend Snippet: MiR-655-3p impedes HCC tumorigenesis via targeting ACER3. (a-k) HCCLM3 and Huh-7 cells were transfected with miR-NC, miR-655-3p, miR-655-3p + vector, or miR-655-3p + ACER3. (a-c) Cell proliferation analysis using CCK-8 and colony formation assays. (d, e) Transwell assay for cell migration and invasion. (f) Tube formation assay for cell angiogenesis analysis. (g-i) Flow cytometry assay for cell cycle distribution and cell apoptosis rate. (j, k) Western blot analysis of the protein levels of E-cadherin, vimentin and N-cadherin. * P < 0.05.

Article Snippet: The primary antibodies included: E-cadherin (ab40772, 1:2000, Abcam), vimentin (ab92547, 1:2000, Abcam), N-cadherin (ab18203, 1:2000, Abcam), ACER3 (1:500, Saierbio, Tianjin, China) and GAPDH (ab181602, 1:1000, Abcam).

Techniques: Transfection, Plasmid Preparation, CCK-8 Assay, Transwell Assay, Migration, Tube Formation Assay, Flow Cytometry, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: How Cancer Cells Invade Bladder Epithelium and Form Tumors: The Mouse Bladder Tumor Model as a Model of Tumor Recurrence in Patients

doi: 10.3390/ijms22126328

Figure Lengend Snippet: Expression of α3β1 integrin and N-cadherin in MB49-GFP cancer cells with internalized nanoparticles in vitro and in vivo. ( A ) Western blot analysis of MB49 cell culture lysates. The detected bands proved that MB49 cells expressed N-cadherin (120 kDa) and α3β1 integrin (117 kDa). β-actin (45 kDa) was used as a loading control. Molecular weight markers are indicated. ( B , D ) GFP-transduced and α3β1 integrin- or N-cadherin-positive cancer cells (arrows) (orange-colored, due to merged green fluorescence of GFP and red fluorescence of α3β1 integrin or N-cadherin) in floating cancer cells (arrows) in the lumen of the bladder 1 hour after intravesical application. ( C , E ) GFP-transduced and α3β1 integrin- or N-cadherin-positive cancer cells (arrows) (orange-colored, due to merged green fluorescence of GFP and red fluorescence of α3β1 integrin or N-cadherin) under the basal lamina 72 h after intravesical application. Note dark spots of no fluorescence inside cancer cells, representing internalized nanoparticles in endosomes (in ( B , D )). Nuclei were stained with DAPI (blue fluorescence). The dashed line represents the basal lamina. U—urothelium; L—lumen of the urinary bladder; CT—connective tissue. Bars are 10 µm.

Article Snippet: N-cadherin , Rabbit polyclonal , 1:200/1:100 , Ab18203 , Abcam.

Techniques: Expressing, In Vitro, In Vivo, Western Blot, Cell Culture, Control, Molecular Weight, Fluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: How Cancer Cells Invade Bladder Epithelium and Form Tumors: The Mouse Bladder Tumor Model as a Model of Tumor Recurrence in Patients

doi: 10.3390/ijms22126328

Figure Lengend Snippet: Intercellular junctions between urothelial cells in tumors developed 3 weeks after intravesical application of MB49-GFP cancer cells. Scanning electron micrograph ( A ) and transmission electron micrograph ( B ) of superficial tumor urothelial cells (TUC) connected by well-developed tight junctions (arrows). ( C ) Strong immunofluorescence reaction against β-catenin (red fluorescence) on boundaries of tumor urothelial cells (green fluorescence due to GFP). Nuclei were stained with DAPI. ( D ) Immunogold labeling of β-catenin (arrowheads) on lateral plasma membranes between two adjacent TUC. ( E ) Pronounced immunofluorescence reaction against desmoplakins 1 and 2 (red fluorescence) on boundaries of tumor urothelial cells (green fluorescence due to GFP). Nuclei were stained with DAPI. ( F ) Ultrastructure of two adjacent TUC connected by desmosome (arrowhead). ( G ) Punctate cytoplasmic and membranous immunofluorescence reaction against N-cadherin (red fluorescence) in tumor urothelial cells (green fluorescence due to GFP). Nuclei were stained with DAPI. ( H ) Immunogold labeling of N-cadherin (arrowheads) on lateral plasma membranes between adjacent TUC. Note heavily enlarged intercellular space (asterisk) between cells. ( I ) Strong punctate cytoplasmic immunofluorescence reaction to α3β1 integrin (red fluorescence) in all tumor urothelial cells (green fluorescence due to GFP). Nuclei were stained with DAPI. Asterisk denotes a macrophage. ( J ) Immunogold labeling of α3β1 integrin (arrowheads) on lateral plasma membranes between adjacent TUC. Enlarged intercellular spaces were present between them (asterisks). L—lumen of the urinary bladder; n—nucleus. Bars are 10 µm ( A , C , E , G , I ), 600 nm ( B , D , H , J ), 300 nm ( F ).

Article Snippet: N-cadherin , Rabbit polyclonal , 1:200/1:100 , Ab18203 , Abcam.

Techniques: Transmission Assay, Immunofluorescence, Fluorescence, Staining, Labeling, Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: How Cancer Cells Invade Bladder Epithelium and Form Tumors: The Mouse Bladder Tumor Model as a Model of Tumor Recurrence in Patients

doi: 10.3390/ijms22126328

Figure Lengend Snippet: Characteristics of a bladder tumor margin 3 weeks after intravesical application of MB49-GFP cancer cells. ( A ) Semi-thin section of a bladder tumor with an easily recognized margin (arrow) between the urothelium (U) and prominent tumor formation (T). Toluidine blue staining. ( B ) Histological section of bladder tumor with a hardly recognized margin (arrow) between the urothelium (U) and sessile tumor formation (T). H&E staining. ( C ) Scanning electron micrograph of a papillary tumor with an outlined tumor margin (boxed area). ( D ) A higher magnification view of the tumor margin (the boxed area from ( C )) with well-developed tight junctions (arrow) between a tumor cell (TC) and a normal urothelial cell (NUC). Note the different structures of the plasma membranes of the two cell types. The tumor cell had a typically ruffled plasma membrane, while the urothelial cell had a characteristic scalloped appearance of the apical plasma membrane. ( E ) The tumor margin (arrow) was revealed due to a positive immunofluorescence reaction against E-cadherin (green fluorescence) among cells of the urothelium (U) and a negative immunofluorescence reaction against E-cadherin among urothelial cells of the tumor formation (T). Note that a weak E-cadherin positivity was present only in deeper urothelial layers of the tumor. ( F ) A higher magnification view (from E ) of the tumor margin (arrow) exposed due to different immunofluorescence patterns of E-cadherin (green fluorescence) in normal NUC than in tumor urothelial cells (TUC). ( G ) The tumor margin (arrow) was revealed due to positive immunofluorescence reaction against desmoplakin 1/2 (red fluorescence) among cells of the urothelium (U) and negative immunofluorescence reaction against desmoplakin 1/2 among cells of tumor formation (T). Note that desmoplakin 1/2 positivity was present only in deeper urothelial layers of the tumor. ( H ) A higher magnification view (from ( G )) of the tumor margin (arrow) exposed due to different immunofluorescence patterns of desmoplakin 1/2 (red fluorescence) in NUC than in TUC. Nuclei were stained with DAPI (blue fluorescence) in ( E – H ). TC—tumor cell; L—lumen of the urinary bladder. Bars are 100 µm ( A ); 20 µm ( B ); 10 µm ( C , E – H ); and 2 µm ( D ).

Article Snippet: N-cadherin , Rabbit polyclonal , 1:200/1:100 , Ab18203 , Abcam.

Techniques: Staining, Clinical Proteomics, Membrane, Immunofluorescence, Fluorescence

Journal: eLife

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.7554/eLife.92068

Figure Lengend Snippet: ( a ) Top panel: Chemical structures of N-cadherin antagonists CRS-066, LCRF-0006, and analogues RB-192 and RB-028. Active side chain or modified side chain depicted by highlighted box. Middle panel: Average cell adhesion index values of preovulatory COCs (11 hr post-hCG) interacting with a fibronectin substrate in presence of vehicle or N-cadherin antagonists CRS-066, LCRF-0006, and side chain modified analogues RB-192 and RB-028 or vehicle at respective doses. Cell indices were determined using the xCELLigence Impedance system over 15 hr ( n = 3 independent experiments with 2 technical replicates per treatment). Bottom panel: Mean ± SD adhesion index values at 6 hr different superscript letters indicate significant differences (P<0.05), and IC 50 values of respective drugs calculated from dose–response curve and compared using one-way ANOVA. ( b, d ) Representative confocal images of N-cadherin adherens junctions on SK-OV-3 cells treated with N-cadherin antagonists CRS-066 (0.1–1.0 μM), LCRF-0006 (36–360 μM), or vehicle for 24 hr ( N = 3). Scale bar: 40 μM. ( c, e ) Quantification of N-cadherin adherens junctions. Mean of total pixel intensity in red channel, ± SEM of at least 50 cells. N = 3 independent experiments. Statistical analyses with two-tailed unpaired t -test. * denotes <0.01. ( f ) Bright-field images of spheroid formation in 67NR mouse mammary cell line expressing ectopic Cdh2 were treated with CRS-066 or vehicle at respective time points. Cells were seeded at 2000 cells per well, and formation of spheroids was assessed by imaging every hour for 6 h. ( g ) Mean ± SEM of spheroid area in 67NR-Cdh2 cells treated with either vehicle or increasing doses of CRS-066 (0–2 μM) over 6 hr.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat# 8480; 1:500), E-cadherin (CST, Cat# 14472; 1:500); a-tubulin (Thermo Fisher; Cat# 236-10501); Ki-67 (CST; Cat# 9449; 1:1000); and Cleaved caspase 3 (CST; Cat# 9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Analogues, Modification, Two Tailed Test, Expressing, Imaging

Journal: eLife

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.7554/eLife.92068

Figure Lengend Snippet: ( a ) Bright-field images of spheroid formation assay in either wild-type 67NR cells lacking endogenous N-cadherin and unable to form spheroids and in 67NRs Cdh2 stably expressing N-cadherin and successfully undergo spheroid compaction within 6 hr. ( b ) Example of automated ‘spheroid mask’ image segmentation used to calculate spheroid area using ImageJ software. ( c ) Mean ± SEM of spheroid area in 67NR-Cdh2 or 67NR WT over 6 hr calculated after ‘spheroid masking’.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat# 8480; 1:500), E-cadherin (CST, Cat# 14472; 1:500); a-tubulin (Thermo Fisher; Cat# 236-10501); Ki-67 (CST; Cat# 9449; 1:1000); and Cleaved caspase 3 (CST; Cat# 9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Tube Formation Assay, Stable Transfection, Expressing, Software

Journal: eLife

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.7554/eLife.92068

Figure Lengend Snippet: ( a–c ) Time-course of Cdh2, ctnnb1, and Cdh1 mRNA expression in isolated granulosa cell (GCs) or cumulus–oocyte complexes (COCs) from mouse ovaries at indicated time points after eCG and hCG stimulation of folliculogenesis and ovulation ( N = 3 animals per time point). Cdh2 and Ctnnb1 levels are high in GC and COC throughout folliculogenesis, with a transient drop in Cdh2 level 12 hr after ovulation stimulus, while E-cadherin was high in COCs and significantly reduced by ovulation stimulus. The levels shown of the indicated mRNAs were determined by TaqMan qPCR normalised to Rpl19. ( d ) Immunofluorescent staining of N-cadherin, β-catenin, and E-cadherin throughout ovarian folliculogenesis. Confocal images of mouse ovarian sections obtained from eCG primed mice and stained using anti N-cadherin (left panel), anti-β-catenin (middle panel), and E-cadherin (right panel). DNA is counterstained with Hoechst. Arrows indicate presence of N-cadherin and β-catenin at granulosa–granulosa cell junctions in secondary and antral follicle stages. Arrowheads indicate presence of N-cadherin and β-catenin at oocyte–cumulus interface. High magnification images show transzonal projections extending from cumulus cells and anchored to oocyte membrane Scale bar: 50 µM. ( e ) Whole-mount immunofluorescent staining showing co-localisation of N-cadherin (green) and E-cadherin (red) at the oocyte plasma membrane in mouse COC from antral follicles of eCG primed mice. N-cadherin is also evident on cumulus cell surfaces and transzonal projections. Cumulus cell and oocyte nuclear DNA are counterstained with Hoechst. Scale bar: 50 µM. ( f ) Whole-mount immunostaining shows loss of β-catenin and E-cadherin at the oocyte plasma membrane after treatment with CRS-066. COCs obtained from antral follicles of eCG primed mice and treated with CRS-066 or vehicle for 4 hr. COCs were fixed and stained with anti-E-cadherin (green) and anti-β-catenin (red). DNA was counterstained with Hoechst. Scale bar: 50 µM.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat# 8480; 1:500), E-cadherin (CST, Cat# 14472; 1:500); a-tubulin (Thermo Fisher; Cat# 236-10501); Ki-67 (CST; Cat# 9449; 1:1000); and Cleaved caspase 3 (CST; Cat# 9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Expressing, Isolation, Staining, Membrane, Clinical Proteomics, Immunostaining

Journal: eLife

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.7554/eLife.92068

Figure Lengend Snippet: PLA confirms close-proximity interaction between N-cadherin and β-catenin (red) on granulosa cell junctions ( a, b ) and at oocyte membrane ( c ).

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat# 8480; 1:500), E-cadherin (CST, Cat# 14472; 1:500); a-tubulin (Thermo Fisher; Cat# 236-10501); Ki-67 (CST; Cat# 9449; 1:1000); and Cleaved caspase 3 (CST; Cat# 9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Membrane

Journal: eLife

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.7554/eLife.92068

Figure Lengend Snippet: COCs from eCG primed mice were treated with LCRF-0006 (36–360 µM) or CRS-066 (0.1–1 µM) during in vitro maturation (IVM) (EGF and FSH stimulated) and cumulus expansion was assessed after 12 hr or gene expression assessed after 10 hr IVM. ( a, d ) Representative bright-field images of COCs after 12 hr IVM treated with LCRF-0006 or CRS-066. Scale bar: 10 µm. ( b, e ) Mean ± SEM of cumulus expansion indices from a and d n = >20 COCs per experiment. N = 4 independent experiments, different superscript letters indicate significant differences (P<0.05), *p < 0.05, **p < 0.01. ( c, f ) Effect of N-cadherin antagonist treatment during IVM (10 hr) on the expression of key genes involved in COC expansion during IVM. Mean ± SEM. N = 3 independent experiments. Statistical testing with one-way ANOVA, *p < 0.05, **p < 0.01. ( g, h ) Gene ontology enrichment of biological pathways and molecular functions of significantly differentially downregulated genes identified in RNA-Seq analysis of COCs after CRS-066 (0.3 μM) treatment compared to vehicle treatment. All data are presented as the ratio of CRS-066 over vehicle ( N = 3). ( I, j ) Gene set enrichment analysis (GSEA) plot demonstrating the upregulation of Ctnnb1 and Hippo signalling pathways in CRS-066- versus vehicle-treated COCs. Net enrichment score (NES) values are shown. N = 3 independent biological replicates. ( k ) Heatmap representing the relative expression profiles of transcripts involved in Wnt\β-catenin, Hippo\YAP, and ovarian signalling axes.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat# 8480; 1:500), E-cadherin (CST, Cat# 14472; 1:500); a-tubulin (Thermo Fisher; Cat# 236-10501); Ki-67 (CST; Cat# 9449; 1:1000); and Cleaved caspase 3 (CST; Cat# 9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: In Vitro, Gene Expression, Expressing, RNA Sequencing

Journal: eLife

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.7554/eLife.92068

Figure Lengend Snippet: ( a ) Dosing schedule for in vivo studies with N-cadherin antagonist CRS-066 (50 mg/kg). ( b ) Principal component analyses of ovaries collected from mice treated with either CRS-066 or vehicle 16 hr post-hCG. ( c ) Volcano plot of differentially expressed genes (DEGs) (adjusted p < 10 –6 and log 2 FC >0.5).

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat# 8480; 1:500), E-cadherin (CST, Cat# 14472; 1:500); a-tubulin (Thermo Fisher; Cat# 236-10501); Ki-67 (CST; Cat# 9449; 1:1000); and Cleaved caspase 3 (CST; Cat# 9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: In Vivo

Journal: eLife

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.7554/eLife.92068

Figure Lengend Snippet: ( a ) Ovulation rate of 21-day-old mice treated with CRS-066 (50 mg/kg) or vehicle (7.5% DMSO in 0.9% saline). Cumulus–oocyte complexes (COCs) in oviducts counted 16 hr after hCG injection. Graph represents mean ± SEM from N = 6 animals; ***p < 0001 (unpaired two-tailed t -test). ( b ) Histology of ovaries by haematoxylin and eosin staining. CL indicates corpus leuteum; arrows indicate trapped oocytes in CL. Scale bar: 100 µm. ( c ) Hierarchical clustering of RNA-sequencing analysis results shows differentially expressed genes between CRS-066- and vehicle-treated mice ( N = 6 mice per treatment). Gene ontology enrichment of biological pathways ( d ) and molecular functions ( e ) of significantly differently downregulated genes in CRS-066-treated mouse ovaries compared to vehicle treated ovaries. ( f, g ) Gene set enrichment analyses (GSEA) plot shows downregulation of Ctnnb1 and Hippo signalling pathways in CRS-066-treated mice ovaries compared to vehicle-treated ovaries. Net enrichment score (NES) values are shown. ( h ) Heatmap representing the relative expression profiles of transcripts involved in Wnt\β-catenin, Hippo\YAP and ovarian signalling axes in CRS-066-treated ovaries compared to vehicle. N = 3 biological replicates. Relative mRNA expression of key genes involved in gonadotrophin signalling and oocyte function ( i ), COC expansion and ovulation ( j ), or folliculogenesis ( k ) hr in ovaries treated with CRS-066 compared to vehicle treatment and determined by quantitative reverse transcription PCR (qRT-PCR). Bar graph show mean ± SEM. N = 6 ovaries from independent CRS or vehicle-treated mice. Statistical testing with Student’s t -test; *p < 0.05; **p < 0.01; ****p < 0.00001. ( l ) Follicle counts at primary, secondary, pre-antral, antral, and ovulatory stages in ovaries from mice treated with either CRS-006 (50 mg/kg) or vehicle control (7.5% DMSO). N = 3 mice/treatment/time point. ( m ) Representative follicle morphology H&E (left) section and N-cadherin immunofluorescence (right) section in mice treated with either CRS-066 or vehicle. H&E and immunofluorescence highlight disorganised granulosa cells organisation. Asterisks indicate loss of transzonal projections between oocyte and cumulus cells. Scale bar: 30 µm. ( n ) Representative confocal immunofluorescent images of mouse ovaries stained with anti-cleaved caspase 3 and anti-Ki-67. Scale bar: 30 µm. ( o ) Relative mRNA expression of key genes involved in oocyte growth and ovulation in CRS-066- or vehicle-treated mice ( N = 3/treatment) at either 44 hr post eCG or 11 hr post hCG.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat# 8480; 1:500), E-cadherin (CST, Cat# 14472; 1:500); a-tubulin (Thermo Fisher; Cat# 236-10501); Ki-67 (CST; Cat# 9449; 1:1000); and Cleaved caspase 3 (CST; Cat# 9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Saline, Injection, Two Tailed Test, Staining, RNA Sequencing, Expressing, Reverse Transcription, Quantitative RT-PCR, Control, Immunofluorescence

Journal: eLife

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.7554/eLife.92068

Figure Lengend Snippet: ( a ) qPCR analysis of relative mRNA expression of Cdh2, Areg, Ptgs2, and Cyp19a1 in ovaries of control ( Cdh2 Fl/+ ; Amhr2 Cre ) and granulosa-specific Cdh2 null mutants ( Cdh2 Fl/Fl ; Amhr2 Cre ), n = 6 individual animals, *p < 0.05, #p < 0.08. ( b–e ) Immunofluorescent analysis of N-cadherin protein in ovaries of control ( Cdh2 Fl/+ ; Amhr2 Cre ) and granulosa-specific Cdh2 null mutants ( Cdh2 Fl/Fl ; Amhr2 Cre ), showing mosaic depletion of N-cadherin in granulosa cells of mutant follicles. Arrows indicate mosaic regions with persistent N-cadherin. ( f ) Ovulation rate of 21-day-old mice with indicated control or granulosa-specific mutant genotypes. Cumulus–oocyte complexes (COCs) in oviducts counted 16 hr after hCG injection. Graph represents mean ± SEM from N = 4 and 7 animals, respectively; **p < 0.01 (unpaired two-tailed t -test). ( g ) Histology of ovaries by haematoxylin and eosin staining. Scale bar: 100 µm.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat# 8480; 1:500), E-cadherin (CST, Cat# 14472; 1:500); a-tubulin (Thermo Fisher; Cat# 236-10501); Ki-67 (CST; Cat# 9449; 1:1000); and Cleaved caspase 3 (CST; Cat# 9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Expressing, Control, Mutagenesis, Injection, Two Tailed Test, Staining

Journal: Scientific Reports

Article Title: UDCA ameliorates inflammation driven EMT by inducing TGR5 dependent SOCS1 expression in mouse macrophages

doi: 10.1038/s41598-024-75516-9

Figure Lengend Snippet: Impact of BAs on activated macrophage-driven EMT of IECs. Murine peritoneal macrophages were treated with 100µM Bile acids of interest and activated by 100ng/ml of LPS. After 24 h of incubation, the culture supernatant was collected. CT26 cells were treated with macrophage conditioned medium. (A) MTT assay was performed to analyze the cell proliferation fold change of macrophage culture medium-treated CT26 cells. (B) Impact on the colony formation ability of supernatant-treated CT26 cells upon BA treatment of macrophages showed that UDCA had the most regulatory effect among all the tested BAs. Quantified histogram representation of relative colony number and the absorbance of stain taken up by the cells. (C) Representative images from in vitro wound healing assays demonstrate that at 48 h, the cell migration of macrophage-conditioned medium-treated CT26 cells was significantly reduced upon UDCA treatment. (D) Representative images showing the results of Transwell invasion assay of CT26 cells treated with macrophage-conditioned medium. Quantified histogram representation of the relative number of cells per field and the absorbance of stain taken up by the cells. (E) Post-translational expression of EMT markers N-Cadherin, Snail, and Slug was analyzed by western blotting. Histograms representing densitometric analysis w.r.t β-Actin. F , G) Pro-inflammatory cytokine expression and secretion by BA-treated activated macrophages were quantified by qPCR and ELISA, respectively. Data represented as mean ± SD of at least triplicate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001. Raw images of western blots are provided as supplementary file.

Article Snippet: The non-specific binding was blocked with 5% skimmed milk in 1X TBST for 1 h, and after thorough washing, probed with primary antibodies of N-CADHERIN (CST, #13116S), SNAIL (CST, #5879S), SLUG (CST, #9585S), and β-ACTIN (Sigma Aldrich, #A3854) overnight at 4 ̊C and HRP conjugated anti-rabbit IgG secondary antibody (Jackson Immuno Research Laboratories, #111-035003) for 4–5 h in 4 ̊C.

Techniques: Incubation, MTT Assay, Staining, In Vitro, Migration, Transwell Invasion Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: UDCA ameliorates inflammation driven EMT by inducing TGR5 dependent SOCS1 expression in mouse macrophages

doi: 10.1038/s41598-024-75516-9

Figure Lengend Snippet: Impact of BAR silencing on activated macrophage-driven EMT of IECs. RAW264.7 Macrophages were transfected with BAR-specific siRNAs. After 6 h of incubation followed by 24-hour rest, the cells were treated with 100µM UDCA and activated by 100ng/ml of LPS. After 24 h of incubation, the culture supernatant was collected. CT26 cells were treated with macrophage conditioned medium. (A) MTT assay was performed to analyze the effect of BA and BAR silencing on supernatant-treated CT26 cells proliferation. (B) Impact was observed on the colony formation of CT26 cells upon TGR5 silencing. Quantified histogram representation of relative colony number and the absorbance of stain taken up by the cells. (C) Representative images from in vitro wound healing assays after 48 h of incubation demonstrated that cell migration significantly reduced on UDCA treatment but was reversed upon TGR5 silencing. (D) Representative images showing the results of transwell invasion assay of CT26 cells treated with TGR5 deficient macrophage-conditioned medium. Quantified histogram representation of the relative number of cells per field and the absorbance of stain taken up by the cells. (E) Western blot analysis of protein level expression of N-Cadherin, Snail, and Slug in TGR5 deficient-macrophage conditioned media-treated CT26 cells after 48 h of incubation. Densitometry w.r.t β-Actin represented as mean ± SD of triplicate experiments. F , G) Pro-inflammatory cytokine expression and secretion by BA-treated and TGR5 deficient macrophages were quantified by qPCR and ELISA, respectively. Data represented as mean ± SD of at least triplicate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000. Raw images of western blots are provided as supplementary files.

Article Snippet: The non-specific binding was blocked with 5% skimmed milk in 1X TBST for 1 h, and after thorough washing, probed with primary antibodies of N-CADHERIN (CST, #13116S), SNAIL (CST, #5879S), SLUG (CST, #9585S), and β-ACTIN (Sigma Aldrich, #A3854) overnight at 4 ̊C and HRP conjugated anti-rabbit IgG secondary antibody (Jackson Immuno Research Laboratories, #111-035003) for 4–5 h in 4 ̊C.

Techniques: Transfection, Incubation, MTT Assay, Staining, In Vitro, Migration, Transwell Invasion Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: UDCA ameliorates inflammation driven EMT by inducing TGR5 dependent SOCS1 expression in mouse macrophages

doi: 10.1038/s41598-024-75516-9

Figure Lengend Snippet: Impact of SOCS1 silencing on activated macrophage-driven EMT of IECs. RAW264.7 Macrophages were transfected with SOCS1 siRNAs. After 6 h of incubation followed by 24-hour rest, the cells were treated with 100µM UDCA and activated by 100ng/ml of LPS. After 24 h of incubation, the culture supernatant was collected. CT26 cells were treated with macrophage conditioned medium. (A) MTT assay was performed to analyze the effect of SOCS1 knockdown in macrophages on activated macrophage culture supernatant-treated CT26 cell proliferation. (B) Impact was observed on the colony formation of CT26 cells upon SOCS1 silencing. The regulator effect of UDCA was significantly reduced in the absence of SOCS1. Quantified histogram representation of relative colony number and the absorbance of stain taken up by the cells. (C) Representative images from in vitro wound healing assays after 48 h of incubation demonstrated that cell migration significantly reduced on UDCA treatment but was reversed upon SOCS1 silencing. (D) Representative images showing the results of trans-well invasion assay of CT26 cells treated with SOCS1 deficient macrophage-conditioned medium. Quantified histogram representation of the relative number of cells per field and the absorbance of stain taken up by the cells. (E) Western blot analysis of N-Cadherin, Snail, and Slug protein level expression in SOCS1 deficient-macrophage conditioned media-treated CT26 cells after 48 h of incubation. Densitometry w.r.t β-Actin F , G) Pro-inflammatory cytokine expression and secretion by BA-treated and SOCS1 knockdown macrophages were quantified by qPCR and ELISA, respectively. Data represented as mean ± SD of at least triplicate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Raw images of western blots are provided as supplementary files.

Article Snippet: The non-specific binding was blocked with 5% skimmed milk in 1X TBST for 1 h, and after thorough washing, probed with primary antibodies of N-CADHERIN (CST, #13116S), SNAIL (CST, #5879S), SLUG (CST, #9585S), and β-ACTIN (Sigma Aldrich, #A3854) overnight at 4 ̊C and HRP conjugated anti-rabbit IgG secondary antibody (Jackson Immuno Research Laboratories, #111-035003) for 4–5 h in 4 ̊C.

Techniques: Transfection, Incubation, MTT Assay, Knockdown, Staining, In Vitro, Migration, Invasion Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: UDCA ameliorates inflammation driven EMT by inducing TGR5 dependent SOCS1 expression in mouse macrophages

doi: 10.1038/s41598-024-75516-9

Figure Lengend Snippet: AOM-DSS-induced colorectal cancer model treated with UDCA powdered diet. (A) The experimental procedure for developing the AOM-DSS-induced CAC model and 0.2% UDCA administration. (B) Representative graph of Percentage of Weight loss 7 days post AOM injection, from the initiation of DSS cycle 1. (C) A representative image of the colon highlights the visual difference in colon length and tumor occurrence. (D) Representative graph showing the occurrence of colon tumors in the groups (E) Tumor incidence % (F) Colon length in cm (G) Colon weight/length ratio (H) H & E Staining of representative histological sections of colons from the groups (200 × magnification). (I) DAI Score was calculated based on the colon morphology, mucosal integrity, crypt integrity, and immune cell infiltration. (J) mRNA expression of SOCS1 K) mRNA expression of pro-inflammatory cytokines like IL- 1β, IL-6, IL-12, and TNF-α L) mRNA expression of mesenchymal markers N-Cadherin, Snail, and Slug M) mRNA expression of TGR5 receptor in colon tissue samples were quantified, and impact of UDCA was inferred. Data represented as mean ± SD of n = 12–13. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The non-specific binding was blocked with 5% skimmed milk in 1X TBST for 1 h, and after thorough washing, probed with primary antibodies of N-CADHERIN (CST, #13116S), SNAIL (CST, #5879S), SLUG (CST, #9585S), and β-ACTIN (Sigma Aldrich, #A3854) overnight at 4 ̊C and HRP conjugated anti-rabbit IgG secondary antibody (Jackson Immuno Research Laboratories, #111-035003) for 4–5 h in 4 ̊C.

Techniques: Injection, Staining, Expressing

Journal: Molecular Cancer

Article Title: Alpha-enolase as a potential cancer prognostic marker promotes cell growth, migration, and invasion in glioma

doi: 10.1186/1476-4598-13-65

Figure Lengend Snippet: Correlation between the clinicopathologic characteristics and expression of HDGF protein in glioma

Article Snippet: Western blot was carried out according as described [ ] with rabbit polyclonal anti-ENO1 antibody (1:1000; Proteintech, USA), anti-NF-κB, Cyclin D1, Cyclin E1 and E2F1 antibody (1:400; Santa Cruz Biotechnology, Santa Cruz, USA), anti-Rb, pRb (Ser,780), Akt, pAkt (Ser473), PI3K, pPI3K (Tyr458), Snail, Slug, β-catenin, E-Cadherin, N-Cadherin and Vimentin antibody (1:1000; Cell Signaling Technology, Danvers, USA).

Techniques: Expressing

Journal: Molecular Cancer

Article Title: Alpha-enolase as a potential cancer prognostic marker promotes cell growth, migration, and invasion in glioma

doi: 10.1186/1476-4598-13-65

Figure Lengend Snippet: Summary of univariate and multivariate Cox regression analysis of overall survival duration

Article Snippet: Western blot was carried out according as described [ ] with rabbit polyclonal anti-ENO1 antibody (1:1000; Proteintech, USA), anti-NF-κB, Cyclin D1, Cyclin E1 and E2F1 antibody (1:400; Santa Cruz Biotechnology, Santa Cruz, USA), anti-Rb, pRb (Ser,780), Akt, pAkt (Ser473), PI3K, pPI3K (Tyr458), Snail, Slug, β-catenin, E-Cadherin, N-Cadherin and Vimentin antibody (1:1000; Cell Signaling Technology, Danvers, USA).

Techniques: Expressing

Journal: Molecular Cancer

Article Title: Alpha-enolase as a potential cancer prognostic marker promotes cell growth, migration, and invasion in glioma

doi: 10.1186/1476-4598-13-65

Figure Lengend Snippet: Kaplan-Meier survival analysis of overall survival duration in 136 glioma patients according to ENO1 protein expression. Accumulation expression of ENO1 was unfavorable for glioma prognosis. The log-rank test was used to calculate P values.

Article Snippet: Western blot was carried out according as described [ ] with rabbit polyclonal anti-ENO1 antibody (1:1000; Proteintech, USA), anti-NF-κB, Cyclin D1, Cyclin E1 and E2F1 antibody (1:400; Santa Cruz Biotechnology, Santa Cruz, USA), anti-Rb, pRb (Ser,780), Akt, pAkt (Ser473), PI3K, pPI3K (Tyr458), Snail, Slug, β-catenin, E-Cadherin, N-Cadherin and Vimentin antibody (1:1000; Cell Signaling Technology, Danvers, USA).

Techniques: Expressing

Journal: Molecular Cancer

Article Title: Alpha-enolase as a potential cancer prognostic marker promotes cell growth, migration, and invasion in glioma

doi: 10.1186/1476-4598-13-65

Figure Lengend Snippet: Effect of shRNA to stably knock down the expression of ENO1 in human glioma cell lines U251 and U87. Different treatments included PLV-Ctr. (A) . RT-PCR shows transcriptional levels of the ENO1 gene with ARF used as a loading control. (B) . Western blot showing protein expression levels in shENO1 and PLV-Ctr treatments. A representative image of three different experiments is shown. β-actin served as a loading control. Bar graph shows the relative expression of protein among the groups. Data are presented as mean ± SD for three independent experiments (* P < 0.05, ** P > 0.05).

Article Snippet: Western blot was carried out according as described [ ] with rabbit polyclonal anti-ENO1 antibody (1:1000; Proteintech, USA), anti-NF-κB, Cyclin D1, Cyclin E1 and E2F1 antibody (1:400; Santa Cruz Biotechnology, Santa Cruz, USA), anti-Rb, pRb (Ser,780), Akt, pAkt (Ser473), PI3K, pPI3K (Tyr458), Snail, Slug, β-catenin, E-Cadherin, N-Cadherin and Vimentin antibody (1:1000; Cell Signaling Technology, Danvers, USA).

Techniques: shRNA, Stable Transfection, Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot

Journal: Molecular Cancer

Article Title: Alpha-enolase as a potential cancer prognostic marker promotes cell growth, migration, and invasion in glioma

doi: 10.1186/1476-4598-13-65

Figure Lengend Snippet: Stably downregulated ENO1 expression suppressed cell proliferation in vitro and tumorigenicity in vivo. (A) . Effect of ENO1 knockdown on U251 and U87 cell proliferation as measured by MTT assay. Absorbance was read at 490 nm with averages from triplicate wells. Data are presented as mean ± SD for three independent experiments. (B) . Transiently reducing the expression of ENO1 by siRNA inhibited cell proliferation in glioma U251 and U87 cells. (C) . In vitro proliferative ability of glioma cells was significantly decreased in ENO1-suppressed cells compared to PLV-Ctr cells by colony formation assay. (D) . When compared with PLV-Ctr, tumorigenicity of shENO1-U25 and shENO1-U87 cells was markedly reduced in vivo (* P < 0.05). (E) . Immunohistochemical (IHC) staining of ENO1 expression in subcutaneous tumors of mice injected with shENO1 and PLV-Ctr cells.

Article Snippet: Western blot was carried out according as described [ ] with rabbit polyclonal anti-ENO1 antibody (1:1000; Proteintech, USA), anti-NF-κB, Cyclin D1, Cyclin E1 and E2F1 antibody (1:400; Santa Cruz Biotechnology, Santa Cruz, USA), anti-Rb, pRb (Ser,780), Akt, pAkt (Ser473), PI3K, pPI3K (Tyr458), Snail, Slug, β-catenin, E-Cadherin, N-Cadherin and Vimentin antibody (1:1000; Cell Signaling Technology, Danvers, USA).

Techniques: Stable Transfection, Expressing, In Vitro, In Vivo, Knockdown, MTT Assay, Colony Assay, Immunohistochemical staining, Immunohistochemistry, Injection

Journal: Molecular Cancer

Article Title: Alpha-enolase as a potential cancer prognostic marker promotes cell growth, migration, and invasion in glioma

doi: 10.1186/1476-4598-13-65

Figure Lengend Snippet: Stably inhibited ENO1 expression decreases cell migration and invasion. (A) . Stably downregulating ENO reduced the migration ability of shENO1-U251 and shENO1-U87 cells in vitro. (B) . Stably suppressed ENO1 reduced in vitro invasion of shENO1-U251 and shENO1-U87 cells. (C) . Transiently downregulated ENO1 dramatically decreased the migration ability of U251 and U87 cells in vitro. (D) . Transiently suppressed ENO1 inhibited in vitro invasion of U251 and U87 cells. Data were presented were presented as mean ± SD for three independent experiments. * P < 0.05, statistically significant difference.

Article Snippet: Western blot was carried out according as described [ ] with rabbit polyclonal anti-ENO1 antibody (1:1000; Proteintech, USA), anti-NF-κB, Cyclin D1, Cyclin E1 and E2F1 antibody (1:400; Santa Cruz Biotechnology, Santa Cruz, USA), anti-Rb, pRb (Ser,780), Akt, pAkt (Ser473), PI3K, pPI3K (Tyr458), Snail, Slug, β-catenin, E-Cadherin, N-Cadherin and Vimentin antibody (1:1000; Cell Signaling Technology, Danvers, USA).

Techniques: Stable Transfection, Expressing, Migration, In Vitro

Journal: Molecular Cancer

Article Title: Alpha-enolase as a potential cancer prognostic marker promotes cell growth, migration, and invasion in glioma

doi: 10.1186/1476-4598-13-65

Figure Lengend Snippet: ENO1 controls the expression of cell cycle and EMT-associated genes in glioma via PI3K/Akt pathway. (A) . Knocking down endogenous ENO1 expression reduced the expression of pRb (Ser 780), NF-κB, and oncogenic cell cycle regulators including Cyclin D1 and Cyclin E1. However, total Rb and E2F1 were not affected. (B) . Suppressing ENO1 expression decreased the expression of EMT-marker genes including Snail, β-catenin, Vimentin, Slug and N-cadherin but enhanced E-cadherin expression. (C) . Reduced ENO1 expression depressed the expression of phos-PI3K, and Akt, but not their total protein levels. (D) . Western blot analyses of E-cadherin, Cyclin D1, p-Rb in glioma U251 and U87 cells after LY294002 treatment. Each experiment was repeated three times.

Article Snippet: Western blot was carried out according as described [ ] with rabbit polyclonal anti-ENO1 antibody (1:1000; Proteintech, USA), anti-NF-κB, Cyclin D1, Cyclin E1 and E2F1 antibody (1:400; Santa Cruz Biotechnology, Santa Cruz, USA), anti-Rb, pRb (Ser,780), Akt, pAkt (Ser473), PI3K, pPI3K (Tyr458), Snail, Slug, β-catenin, E-Cadherin, N-Cadherin and Vimentin antibody (1:1000; Cell Signaling Technology, Danvers, USA).

Techniques: Expressing, Marker, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Mefunidone Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fphar.2021.713572

Figure Lengend Snippet: Mefunidone inhibited epithelial-to-mesenchymal transition both in vivo and in vitro (A–D) : Western blotting (A) and quantitative analysis of E-cadherin (B) , Snail (C) and vimentin (D) in lung tissues from different groups (E–H) : Western blotting (E) and quantitative analysis of E-cadherin (F) , Snail (G) and vimentin (H) in MLE-12 cells treated with bleomycin (400 μg/ml) with/without mefunidone (40 μg/ml) pretreatment. All experiments were conducted with more than three independent replications. Data were presented as mean ± SD. ANOVA was used for comparison in (B–D) and (F–H) . * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Membranes were blocked and then incubated overnight at 4°C with primary antibodies from the following sources: Fn (Abcam, USA), α-SMA (Sigma, USA), collagen-I (Abcam, USA), Bax (CST, USA), Bcl-2 (CST, USA), cleaved-caspase 3 (Proteintech, USA), E-cadherin (Proteintech, USA), Snail (Proteintech, USA), vimentin (Proteintech, USA), p -ERK1/2 (CST, USA), ERK1/2 (CST, USA), p -Smad2 (CST, USA), α-tubulin (Proteintech, USA), GAPDH (Proteintech, USA), and TGF-β (Abcam, USA).

Techniques: In Vivo, In Vitro, Western Blot, Comparison