Journal: World Journal of Gastrointestinal Surgery

Article Title: Research progress on O-GlcNAcylation in the occurrence, development, and treatment of colorectal cancer

doi: 10.4240/wjgs.v13.i2.96

Figure Lengend Snippet: Summary of Studies on O-GlcNAcylation and colorectal cancer in recent 5 years

Article Snippet: Harosh-Davidovich et al [ ] , 2018 , CT26 murine colon carcinoma cells and NIH-3T3 murine fibroblasts , ATCC , (1) Protein extraction. β-Catenin IP. (2) Affinity purification of β-catenin with Wheat Germ Agglutinin (WGA). (3) WB. (4) Cell motility assay. (5) OGA and OGT silencing. (6) Luciferase reporter assays. (7) qRT-PCR. And (8) In vivo orthotopic mouse model of CRC , O-GlcNAcylation may enhance the proliferation and metastasis of CRC by regulating the expression of catenin and E-cadherin, which proves the influence of O-GlcNAcylation on the poor prognosis of CRC patients.

Techniques: Glycoproteomics, Quantitative Proteomics, Plasmid Preparation, Transfection, Isolation, Purification, RNA Extraction, Protein Extraction, Western Blot, Flow Cytometry, Biomarker Discovery, Expressing, Migration, Immunofluorescence, Confocal Microscopy, Invasion Assay, Co-culture Assay, Gene Expression, Chromatin Immunoprecipitation, Construct, CRISPR, Knock-Out, Knockdown, shRNA, Marker, Activation Assay, Labeling, Microarray, Immunohistochemistry-IF, Activity Assay, Soft Agar Assay, Ligation, In Vitro, Cell Culture, Stable Transfection, CCK-8 Assay, Virus, Reporter Assay, Reverse Transcription, Viability Assay, Colony Assay, Cell Migration Assay, Infection, Microscopy, Mutagenesis, Immunohistochemical staining, Staining, Sequencing, DNA Methylation Assay, Mass Spectrometry, Multiplex Assay, Immunohistochemistry, Proteomic Assay, Affinity Purification, Motility Assay, Luciferase, In Vivo, Fluorescence, In Situ Hybridization, Sample Prep, Concentration Assay, Chemotaxis Assay, Extraction, Bacteria, Adjuvant, Histopathology, Cell Adhesion Assay, Colorimetric Assay

Journal: The Journal of pediatrics

Article Title: Whole exome sequencing reveals a novel mutation in CUL7 in a patient with an undiagnosed growth disorder

doi: 10.1016/j.jpeds.2012.07.055

Figure Lengend Snippet: Proband’s Diagnostic Genetic Testing

Article Snippet: Whole exome sequencing of the proband, his brother, and both parents was performed at the Broad Institute.

Techniques: Diagnostic Assay, Microarray, Sequencing, Methylation

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Casz1 expression pattern during mouse embryogenesis. A, cartoon of the Casz1 gene trap that inserted with a βgeo reporter after Casz1 exon 9 resulting in a truncated Casz1. B, genotyping of Casz1-trapped mice by RT-PCR. C, whole mount X-gal staining (blue) of E9.5 Casz1+/βgeo embryos shows that Casz1 was expressed in the hindbrain, neural tube, and heart, which are further demonstrated by dissected embryo sagittal sections 1, 2, and 3 (100× magnification). Sagittal section 3b (200× magnification) shows that Casz1 was expressed in the cardiomyocytes. D and E, whole mount X-gal staining of E12.5 Casz1+/+, Casz1+/βgeo, and Casz1βgeo/βgeo embryos showed that Casz1 is expressed in the eye, dorsomedial telencephalon, cranial ganglia, nasal placode, somite, neural tube, and heart. F, real time PCR to detect wild type Casz1 allele and Gata4 mRNA levels in E12.5 hearts. G, the protein levels of Casz1a/Casz1b and GAPDH were visualized by immunoblotting E14.5 whole heart lysate with anti-Casz1 antibody and anti-GAPDH antibody. H, whole mount anti-β-galactosidase staining (red) of E14.5 Casz1+/+, Casz1+/βgeo, and Casz1βgeo/βgeo hearts.

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Hypoplasia in the myocardium of Casz1βgeo/βgeo heart. A, phenotype of Casz1+/+, Casz1+/βgeo and Casz1βgeo/βgeo E 15.5 embryos. The edema was seen at the neck region on the back of Casz1βgeo/βgeo embryos but not other embryos (green arrow). B–D, column 1, H&E staining of transverse sections of E15.5 Casz1+/+ (B1), Casz1+/βgeo (C1), and Casz1βgeo/βgeo (D1) embryos. Column 2, right ventricle (RV) magnification of the respective green squares from column 1. Column 3, septum (SP) magnification of respective yellow squares from column 1. Column 4, left ventricle (LV) magnification of respective blue squares from column 1. E, representative photomicrographs indicate accumulation of blood cells in the liver of the Casz1βgeo/βgeo embryo (right panel) but not in the Casz1+/+ (left panel) or Casz1+/βgeo (middle panel) embryo.

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Decreased proliferation in Casz1βgeo/βgeo heart. A, the E14.5 Casz1βgeo/βgeo hearts were morphologically different compared with the Casz1+/βgeo or wild type heart (compare the green arrow targeted region). B, lower magnification of H&E staining of transverse sections from E14.5 embryos (upper panels). The lower panels represent a magnification of the regions that are depicted in the blue squares in the upper panel. The green arrow denotes the septal defect apparent in the serial sectioning of the Casz1βgeo/βgeo heart compared with the Casz1+/+ heart. C, paraffin sections from E14.5 hearts from Casz1+/+ and Casz1βgeo/βgeo hearts were immunostained with antibodies for MF20 (red) and the mitosis marker phosphohistone H3 (pH3, green). The nuclei were stained with DAPI (blue; left top panels, original overview; left bottom panels, high magnification). In the high magnification images, arrows indicate pH3+ cells. The graph (right panel) represents the relative percentage of pH3 positive cells compared with the total number of DAPI positive Casz1βgeo/βgeo or Casz1+/+ cardiomyocytes from at least five sections from each embryo. The Casz1βgeo/βgeo cardiomyocytes proliferate significantly slower than Casz1+/+ cardiomyocytes (p < 0.00001). D, tunnel staining (green) was performed using E14.5 heart paraffin sections (upper panels), and nuclei were stained with DAPI (blue). The lower panels represent magnified areas delineated in the red squares in the upper panels. There was no significant difference in the number of apoptotic cells observed in Casz1βgeo/βgeo cardiomyocytes compared with Casz1+/+ cardiomyocytes.

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Staining, Marker

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Disorganized fiber orientation in Casz1βgeo/βgeo heart. To visualize thin filaments, Casz1+/+ and Casz1βgeo/βgeo E14.5 hearts were stained with phalloidin-488 (green) to detect F-Actin and co-stained with TOPRO3 to show cell nuclei. The left panels show the co-staining results of the left ventricles of the hearts. The right panels represent magnified areas delineated in the red squares in the left panels. Enlarged images showed complete disruption of the fiber orientation/cell alignment in the left ventricle (LV) of Casz1βgeo/βgeo heart.

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Abnormal gene expression in Casz1βgeo/βgeo heart. A, microarray analysis of RNA from three E12.5 Casz1+/+ hearts and three E12.5 Casz1βgeo/βgeo hearts. The heat map was generated using Partek software. Two-thirds of these genes are aberrantly up-regulated, and one-third are aberrantly down regulated in Casz1βgeo/βgeo hearts. B, Partek gene ontology oncology analysis showed that the top two categories of enriched genes are “signaling” and the process of biological adhesion. Biological adhesion genes in Casz1βgeo/βgeo hearts that are up-regulated (red) or down-regulated (blue) are listed below. C, IPA assays showed gene enrichment in the categories of physiological system development and function (panel 1) with a key subcategory being skeletal and muscular system development and function genes that are up-regulated (red) or down-regulated (blue) in Casz1βgeo/βgeo hearts listed below and molecular and cellular function (panel 2) with a key subcategory of genes in molecular transport and the ion transport genes that are up-regulated (red) or down-regulated (blue) listed below. D, verification of microarray results. The mRNA levels of representative genes encoding cell adhesion molecules, muscle contraction, and muscular development proteins and ion channels were evaluated by real time PCR and normalized to GAPDH gene. The mRNA levels of these genes in Casz1βgeo/βgeo hearts were significantly different compared with their levels in Casz1+/+ hearts. The bar graph represents means ± S.D. (all p < 0.005).

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Expressing, Microarray, Generated, Software, Cell Function Assay, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Abnormal expression of genes involved in cell cycle and regulation of cell proliferation in Casz1βgeo/βgeo heart. A, IPA assays showed gene enrichment in the categories of cell cycle and cellular growth and proliferation. The genes that are up-regulated (red) or down-regulated (blue) in Casz1βgeo/βgeo hearts were listed below. B, verification of microarray results. The mRNA levels of representative genes were evaluated by real time PCR and normalized to GAPDH gene. The mRNA levels of these genes in Casz1βgeo/βgeo hearts were significantly different compared with their levels in Casz1+/+ hearts. The bar graph represents means ± S.D. (all p < 0.05).

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Expressing, Microarray, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Validation of Casz1 target genes in cellular models. A, human cardiac fibroblasts were transfected with empty vector (EMV) or CASZ1b vector and cultured for 5 days. Panel a, relative CASZ1b mRNA level was evaluated by real time PCR using human CASZ1 primer. The bar graph represents means ± S.E.(#, p < 0.05). Panel b, Western blot to show the overexpression of Casz1b in CASZ1b overexpressed cells. Panel c, after overexpression of CASZ1b, relative mRNA levels of representative genes were evaluated by real time PCR. The bar graph represents means ± S.E. (#, p < 0.05). B, knockdown or overexpression of Casz1 in HL-1 cells. Panel a, HL-1 cells were transfected with nontargeting control (con) siRNA or Casz1 siRNA, and after a 2-day culture, the relative mRNA levels of representative genes were evaluated by real time. The bar graph represents means ± S.D. (*, p < 0.005). Panel b, HL-1 cells were transfected with EMV or CASZ1b vector, and after a 2-day culture, the relative CASZ1b mRNA levels were evaluated by real time PCR using human CASZ1 primers. The graph represents mean ± S.D. (*, p < 0.005). Panel c, after transfection with CASZ1b and a two-dimensional culture, the relative mRNA levels of representative genes were evaluated by real time PCR. The bar graph represents means ± S.D. (#, p < 0.05; ns represents no statistic significant difference).

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Transfection, Plasmid Preparation, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Sarcomeric organization in Casz1βgeo/βgeo heart. A, GSEA assay indicated the negative enrichment of genes encoding contractile fiber proteins (panels a and b), as well as genes encoding contractile fiber part proteins (panel c). NES, normalized enrichment score; Nom, nominal; FDR, false discovery rate. B, sections containing the trabecular regions of E14.5 hearts were labeled for desmin (red), and nuclei were stained with DAPI (blue). There is a striated pattern for desmin labeling in both wild type and Casz1-deficient hearts, but the striated patterns are not as uniform or apparent in the Casz1-deficient heart.

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Labeling, Staining

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development *

doi: 10.1074/jbc.M114.570416

Figure Lengend Snippet: Abnormal Z line organization in E14.5 Casz1βgeo/βgeo heart. A, cardiomyocytes isolated from Casz1+/+ and Casz1βgeo/βgeo E14.5 embryo hearts were cultured in vitro for 2 days and immunostained for α-actinin (red) and F-actin (green), whereas the nuclei were stained with DAPI (blue). Representative images show that clear Z lines were presented in the wild type cardiomyocytes, but not in the Casz1βgeo/βgeo cardiomyocytes. B, the CHITEST histogram shows the percentage of Casz1+/+ or Casz1βgeo/βgeo cardiomyocytes with clear Z lines (Z line+) or abnormal Z lines (Z line−) (p < 0.00001). The data are from two independent experiments (>100 Casz1+/+ or Casz1βgeo/βgeo cardiomyocytes were counted in each experiment). C, more representative images show that clear Z lines are present in the Casz1+/+ cardiomyocytes. There are no clear Z lines in these Casz1βgeo/βgeo cardiomyocytes, and the α-actinin staining pattern is distinct compared with the Casz1+/+ cardiomyocytes.

Article Snippet: Rabbit anti-GAPDH (Santa Cruz, 1:4000) and rabbit anti-Casz1 (Rockland, 1:1000) were used to detect GAPDH and Casz1 protein level in heart.

Techniques: Isolation, Cell Culture, In Vitro, Staining

Journal: Nature Communications

Article Title: TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation

doi: 10.1038/s41467-020-17802-4

Figure Lengend Snippet: a – e TMBIM6 expression was analyzed using the GEO database from NCBI. Fibrosarcoma (GSE2719; normal n = 3; tumor n = 7), cervix (GSE63678; cervical normal n = 5; tumor n = 5; endometrial normal n = 5; tumor n = 7; vulvar normal n = 7; tumor n = 6), breast (GSE31448; normal n = 31; basal n = 98; luminal A n = 89; luminal B n = 49; ERBB2 n = 25), lung (GSE19804; normal n = 60; tumor n = 60), and prostate (GSE69223; normal n = 15; tumor n = 15) datasets are presented. Center line of the box represents median; box bounds represent 25th and 75th percentiles; whiskers represent minimum and maximum values. f Representative immunohistochemical staining of TMBIM6 on tissue microarrays containing fibrosarcoma, cervix, breast, lung, and prostate tissue and adjacent normal tissues. TMBIM6 WT and KO HT1080 cells were used as a control for validation of the method. Right; quantification data of TMBIM6 expression (fibrosarcoma normal n = 9; tumor n = 8; cervix normal n = 20; tumor n = 80; breast normal n = 6; tumor n = 97; lung normal n = 15; tumor n = 75; prostate normal n = 32; tumor n = 160). Scale bar, 100 μm. (brown: positive antibody staining, blue: hematoxylin for nuclear staining). Data are presented as means ± SD. **** p < 0.0001, two-tailed unpaired t -test. g Kaplan–Meier curves showing the overall survival analysis in patients with high and low expression of TMBIM6 using GEPIA2 tool. P value with log-rank analysis. BRCA breast invasive carcinoma, CESC cervical squamous cell carcinoma and endocervical adenocarcinoma, SARC sarcoma, LUAD lung adenocarcinoma. h Differentially expressed genes by microarray analysis of mRNA expression levels in TMBIM6 KO and WT HT1080 cells. i Significant ratios in TMBIM6 KO and WT HT1080 cells determined by Gene Ontology analysis. j The graph indicates significant differences in downregulation and upregulation of the indicated category genes in the TMBIM6 KO cells compared with those in TMBIM6 WT cells.

Article Snippet: For establishment of stable cell lines of human fibrosarcoma cells (HT1080) expressing pLenti CMV/TO puro DEST (670-1)-TMBIM6-GCaMP3 (TMBIM6-GCaMP3), cells were incubated with 8 μg/mL of Polybrene (Santa Cruz Biotechnology) and lentiviral particles harboring each gene followed by selection with puromycin dihydrochloride (Santa Cruz Biotechnology) for 1 week.

Techniques: Expressing, Immunohistochemical staining, Staining, Control, Biomarker Discovery, Two Tailed Test, Microarray

Journal: Nature Communications

Article Title: TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation

doi: 10.1038/s41467-020-17802-4

Figure Lengend Snippet: a Proliferation of TMBIM6 KO and WT HT1080 cells, HeLa cells, and MEFs ( n = 3 independent experiments). Data are presented as means ± SD. ** p < 0.01, *** p < 0.001, two-way ANOVA followed by Bonferroni’s post hoc test. b Images of migrated cells in TMBIM6 KO and WT HT1080. Right, quantification of WT cells normalized to KO cells ( n = 6 independent experiments). Scale bars, 100 μm. Data are presented as means ± SD. **** p < 0.0001, two-tailed unpaired t -test. c Images of invasive cells in TMBIM6 KO and WT HT1080. Right, quantification of WT cells normalized to KO cells ( n = 5 independent experiments). Scale bars, 100 μm. Data are presented as means ± SD. **** p < 0.001, two-tailed unpaired t -test. d – f Tumor volume, weight, and size derived from TMBIM6 KO or WT HT1080 cells injected into the flank of 6-week-old nude mice ( n = 6 mice per group). Data are presented as means ± SD. * p < 0.05; ** p < 0.01; **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test in D and two-tailed unpaired t -test in ( e ). g Histological images by immunohistochemical detection of Ki-67. Right, quantification of Ki-67-positive cells in xenograft tumors derived from TMBIM6 KO and WT HT1080 cells ( n = 6 mice per group). Scale bars, 100 μm. Data are presented as means ± SD. **** p < 0.0001, two-tailed unpaired t -test. h – j Tumor volume, weight, and size derived from TMBIM6 KO or WT HeLa cells injected into the flank of 6-week-old nude mice ( n = 6 mice per group). Data are presented as means ± SD. *** p < 0.001; **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test in ( h ) and one-way ANOVA followed by Tukey’s post hoc test in ( i ). k Histological images by immunohistochemical detection of Ki-67. Right, quantification of Ki-67-positive cells in xenograft tumors derived from TMBIM6 KO and WT HeLa cells ( n = 6 mice per group). Scale bars, 100 μm. Data are presented as means ± SD. **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: For establishment of stable cell lines of human fibrosarcoma cells (HT1080) expressing pLenti CMV/TO puro DEST (670-1)-TMBIM6-GCaMP3 (TMBIM6-GCaMP3), cells were incubated with 8 μg/mL of Polybrene (Santa Cruz Biotechnology) and lentiviral particles harboring each gene followed by selection with puromycin dihydrochloride (Santa Cruz Biotechnology) for 1 week.

Techniques: Two Tailed Test, Derivative Assay, Injection, Immunohistochemical staining

Journal: Nature Communications

Article Title: TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation

doi: 10.1038/s41467-020-17802-4

Figure Lengend Snippet: a Expression and phosphorylation of 43 proteins were examined by Proteome Profile Human Phospho-Kinase Array in WT and TMBIM6 KO HT1080 cells. Right; the relative phosphorylations of indicated proteins were quantitated by ImagJ. Blots represent one of two experiments, with similar results obtained. b pAKT, pTSC2, and pNDRG1 were analyzed in TMBIM6 KO and WT HT1080 cells using Western blots (left) and normalized to total proteins of WT cells (right; n = 5 independent experiments). Data are presented as means ± SD. *** p < 0.001, **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test. c Immunoblotting, RT-PCR, and its gene quantification analysis were performed in TMBIM6-HA-stably expressing or non-expressing TMBIM6 KO cells ( n = 3 independent experiments). Data are presented as means ± SD. * p < 0.05, ** p < 0.01, two-way ANOVA followed by Bonferroni’s post hoc test. d After serum starvation for 12 h, TMBIM6 KO and WT HT1080 cells were stimulated with insulin (100 ng/ml), IGF1 (100 ng/ml), or EGF (100 ng/ml), and immunoblotting was performed with indicated antibodies. e Gel filtration assay of extracts of TMBIM6 KO and WT MEFs. The red line represents the size marker. f PLA between indicated proteins (red dots) in TMBIM6 KO and WT HT1080 cells. For the PLA, the ribosomal protein S6 kinase beta-1 (S6K1) was also applied as a negative control. Scale bar, 15 μm. Right, quantification of red dots ( n = 5 independent experiments). Data are presented as means ± SD. * p < 0.05, ** p < 0.01, two-way ANOVA followed by Bonferroni’s post hoc test. g Immunoblot analysis of anti-RPL19 immunoprecipitate (IP) and whole cell lysate (WCL) of TMBIM6 KO and WT HT1080 cells.

Article Snippet: For establishment of stable cell lines of human fibrosarcoma cells (HT1080) expressing pLenti CMV/TO puro DEST (670-1)-TMBIM6-GCaMP3 (TMBIM6-GCaMP3), cells were incubated with 8 μg/mL of Polybrene (Santa Cruz Biotechnology) and lentiviral particles harboring each gene followed by selection with puromycin dihydrochloride (Santa Cruz Biotechnology) for 1 week.

Techniques: Expressing, Phospho-proteomics, Western Blot, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Filtration, Marker, Negative Control

Journal: Nature Communications

Article Title: TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation

doi: 10.1038/s41467-020-17802-4

Figure Lengend Snippet: a mRNA levels of glycolysis- and PPP-related genes in TMBIM6 KO and WT HT1080 cells, as determined by qRT-PCR. Quantification data represent the expression level of genes in the KO cells compared with those in normalized WT cells ( n = 3 independent experiments). Data are presented as means ± SD. **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test. b , c Glucose consumption and lactate production in TMBIM6 KO and WT HT1080 cells ( n = 3 independent experiments). Data are presented as means ± SD. ** p < 0.01; *** p < 0.001, one-way ANOVA followed by Tukey’s post hoc test. d Metabolite analysis in TMBIM6 KO and WT HT1080 cells ( n = 2 independent experiments). e , f mRNA levels of GSH biosynthesis genes and de novo lipid biosynthesis genes in TMBIM6 KO and WT HT1080 cells, as determined by qRT-PCR. Quantification data represent the expression level of genes in the KO cells compared with those in normalized WT cells ( n = 3 independent experiments). Data are presented as means ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test. g Immunofluorescence images of protein synthesis in TMBIM6 KO and WT HT1080 cells. Right, quantification data represent the expression intensity compared with that in normalized WT cells ( n = 3 independent experiments). Data are presented as means ± SD. * p < 0.05, two-tailed unpaired t -test. Scale bar, 15 μm. h Glycosylation-related gene lists in TMBIM6 KO and WT HT1080 cells by microarray. i Glycosylated protein levels in TMBIM6 KO and WT HT1080 cells ( n = 3 independent experiments). Data are presented as means ± SD. * p < 0.05, two-tailed unpaired t -test.

Article Snippet: For establishment of stable cell lines of human fibrosarcoma cells (HT1080) expressing pLenti CMV/TO puro DEST (670-1)-TMBIM6-GCaMP3 (TMBIM6-GCaMP3), cells were incubated with 8 μg/mL of Polybrene (Santa Cruz Biotechnology) and lentiviral particles harboring each gene followed by selection with puromycin dihydrochloride (Santa Cruz Biotechnology) for 1 week.

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Two Tailed Test, Glycoproteomics, Microarray

Journal: Nature Communications

Article Title: TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation

doi: 10.1038/s41467-020-17802-4

Figure Lengend Snippet: a Gel filtration assay of lysates of TMBIM6 KO HT1080 cells transiently overexpressing TMBIM6-HA. b Anti-RICTOR immunoprecipitate (IP) of pooled fractions of TMBIM6 KO HT1080 cells transiently overexpressing TMBIM6-HA. The inputs were analyzed by immunoblotting. c Immunoblot analysis of anti-HA IP and whole cell lysate (WCL) of TMBIM6-HA overexpressing HeLa cells. d PLA between TMBIM6-HA and mTORC2 components (red dots) in TMBIM6 stably overexpressing HT1080 cells. Right, quantification of red dots ( n = 5 independent experiments). Scale bar, 15 μm. Data are presented as means ± SD. *** p < 0.001, **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test. e GST pull-down assay between GST-TMBIM6 and myc-RICTOR. f GST pull-down assay in the presence or absence of HA-TMBIM6. g Immunoblot analysis of WCL of HT1080 cells stably expressing TMBIM6 and transfected with scrambled, mTOR, RICTOR, or SIN1 siRNA and immunoprecipitated with anti-HA antibody. h Immunoblot analysis of the immunoprecipitates with anti-RICTOR antibody and whole cell lysates of HT1080 cells transiently transfected with TMBIM6 and TMBIM6 mutant constructs. i Immunoblot analysis of the immunoprecipitates with anti-HA antibody and input of HT1080 cells transiently transfected with TMBIM6 and TMBIM6 mutant constructs.

Article Snippet: For establishment of stable cell lines of human fibrosarcoma cells (HT1080) expressing pLenti CMV/TO puro DEST (670-1)-TMBIM6-GCaMP3 (TMBIM6-GCaMP3), cells were incubated with 8 μg/mL of Polybrene (Santa Cruz Biotechnology) and lentiviral particles harboring each gene followed by selection with puromycin dihydrochloride (Santa Cruz Biotechnology) for 1 week.

Techniques: Filtration, Western Blot, Stable Transfection, Pull Down Assay, Expressing, Transfection, Immunoprecipitation, Mutagenesis, Construct

Journal: Nature Communications

Article Title: TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation

doi: 10.1038/s41467-020-17802-4

Figure Lengend Snippet: a Immunofluorescence images and fluorescence intensity (right) of TMBIM6-GCaMP3 and TMBIM6 D213A-GCaMP3 in the presence or absence of 10 µM BAPTA-AM. ( n = 5 independent experiments). Scale bar, 15 μm. Data are presented as means ± SD. ** p < 0.01, *** p < 0.001, two-way ANOVA followed by Bonferroni’s post hoc test. b PLA between Rictor and mTOR or between Rictor and RPL19 (red dots) in empty vector, TMBIM6, or D213A-transfected HT108 cells. ( n = 3 independent experiments). Scale bar, 15 μm. Data are presented as means ± SD. **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test. c Immunoblot analysis of the immunoprecipitates with Anti-HA antibody and input of cell lysates with the indicated antibodies. d Immunoblotting and quantification of phosphorylation of AKT in TMBIM6 KO HT1080 cells transfected with empty vector, TMBIM6 WT, and TMBIM6 D213A. ( n = 3 independent experiments). Data are presented as means ± SD. ** p < 0.01, *** p < 0.001, one-way ANOVA followed by Tukey’s p ost hoc test. e Immunofluorescence images and quantification of AKT phosphorylation ( n = 3 independent experiments, total of nine images). Scale bar, 15 μm. Data are presented as means ± SD. **** p < 0.0001, one-way ANOVA followed by Tukey’s post hoc test. f Proliferation analysis of empty vector, TMBIM6, and TMBIM6 D213A-expressed HT1080 cells ( n = 3 independent experiments). Data are presented as means ± SD. ** p < 0.01, **** p < 0.0001, two-way ANOVA followed by Bonferroni’s p ost hoc test. g The quantification analysis of mRNA levels of glycolysis- and PPP-related genes in empty vector, TMBIM6, and TMBIM6 D213A-rescued TMBIM6 KO HT1080 cells, as determined by qRT-PCR. Quantification data represent the expression level of genes compared with those in normalized WT HT1080 cells (red line, n = 3 independent experiments). Data are presented as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, two-way ANOVA followed by Bonferroni’s post hoc test. h , i Glucose consumption and lactate production in empty vector, TMBIM6, and TMBIM6 D213A-rescued TMBIM6 KO HT1080 cells ( n = 3 independent experiments). Data are presented as means ± SD. * p < 0.05, ** p < 0.01, one-way ANOVA followed by Tukey’s post hoc test. j Metabolite analysis in empty vector, TMBIM6, and TMBIM6 D213A-rescued TMBIM6 KO HT1080 cells. Quantification data represent the metabolite level compared with those in empty vector-rescued TMBIM6 KO HT1080 cells ( n = 2 independent experiments).

Article Snippet: For establishment of stable cell lines of human fibrosarcoma cells (HT1080) expressing pLenti CMV/TO puro DEST (670-1)-TMBIM6-GCaMP3 (TMBIM6-GCaMP3), cells were incubated with 8 μg/mL of Polybrene (Santa Cruz Biotechnology) and lentiviral particles harboring each gene followed by selection with puromycin dihydrochloride (Santa Cruz Biotechnology) for 1 week.

Techniques: Immunofluorescence, Fluorescence, Plasmid Preparation, Transfection, Western Blot, Phospho-proteomics, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation

doi: 10.1038/s41467-020-17802-4

Figure Lengend Snippet: a Proliferation of TMBIM6 WT HT1080, TMBIM6 KO HT1080, MCF7, MDA-MB-231, and SKBR3 cells treated with BIA ( n = 3 independent experiments). Data are presented as means ± SD. b Gel filtration assay of HT1080 cells treated with 1.0 μM BIA. The red line represents the size marker. c Immunoblot analysis of anti-HA immunoprecipitate (IP) and whole cell lysate (WCL) of HT1080 cells transiently overexpressing TMBIM6-HA and treated with BIA. d Immunoblotting of p-AKT, AKT, and actin in the indicated cell lines following treatment with BIA. e PLA between TMBIM6-HA and mTORC2 components or between TMBIM6-HA and RPL19 (red dots) in BIA-treated or non-treated TMBIM6 stably overexpressing HT1080 cells. Bottom, quantification of red dots ( n = 5 independent experiments). Scale bar, 20 μm. Data are presented as means ± SD. ** p < 0.01, **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test. f , g Real-time lapse images of HT1080 cells stably expressing TMBIM6-GCaMP3 and G-CEPIAer treated with BIA. Right, mean green intensity of every cells normalized to untreated cells ( n = 5 independent experiments, total of 20 cells for TMBIM6-GCaMP3; total of 16 cells for G-CEPIAer). Scale bar, 15 μm. Data are presented as means ± SD.

Article Snippet: For establishment of stable cell lines of human fibrosarcoma cells (HT1080) expressing pLenti CMV/TO puro DEST (670-1)-TMBIM6-GCaMP3 (TMBIM6-GCaMP3), cells were incubated with 8 μg/mL of Polybrene (Santa Cruz Biotechnology) and lentiviral particles harboring each gene followed by selection with puromycin dihydrochloride (Santa Cruz Biotechnology) for 1 week.

Techniques: Filtration, Marker, Western Blot, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation

doi: 10.1038/s41467-020-17802-4

Figure Lengend Snippet: a Images of migrated cells in HT1080, MCF7, MDA-MB-231, and SKBR3 cells treated with 2.0 μM BIA. Right, quantification of migrated cells in the BIA-treated cells normalized to control cells ( n = 3 independent experiments). Data are presented as means ± SD. ** p < 0.01, *** p < 0.001, two-tailed unpaired t -test. Scale bar, 15 μm. b Images of invasive cells in HT1080 and MBA-MB-231 cells treated with 2.0 μM BIA. Right, quantification of invasive cells in BIA-treated cells normalized to control cells ( n = 3 independent experiments). Data are presented as means ± SD. ** p < 0.01, *** p < 0.001, two-tailed unpaired t -test. Scale bar, 15 μm. c DiI fluorescence images of the indicated cell lines after 7 days of 3D culture. Right, quantification of the intensity of fluorescence in the BIA-treated cells normalized to control cells ( n = 3 independent experiments). Scale bar, 15 μm. Data are presented as means ± SD. *** p < 0.001, **** p < 0.0001, two-tailed unpaired t -test. d Images of control and BIA-treated zebrafish after injection of indicated cell lines into embryos. White and red circles represent cell injection and migration sites, respectively. Images represent one of nine experiments, with similar results obtained. Scale bar, 100 μm. e , f Volume and weight of tumors derived from HT1080 cells injected into nude mice treated with vehicle or 1 mg/kg BIA (vehicle; n = 9, BIA; n = 11 mice). Data are presented as means ± SD. * p < 0.05, *** p < 0.001, **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test in ( e ) and two-tailed unpaired t -test in ( f ). g , h Volume and weight of tumors derived from MDA-MB-231 cells injected into nude mice treated with vehicle or 1 mg/kg BIA ( n = 5 mice per group). Data are presented as means ± SD. Data are presented as means ± SD. *** p < 0.001, **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test in ( g ) and two-tailed unpaired t -test in ( h ).

Article Snippet: For establishment of stable cell lines of human fibrosarcoma cells (HT1080) expressing pLenti CMV/TO puro DEST (670-1)-TMBIM6-GCaMP3 (TMBIM6-GCaMP3), cells were incubated with 8 μg/mL of Polybrene (Santa Cruz Biotechnology) and lentiviral particles harboring each gene followed by selection with puromycin dihydrochloride (Santa Cruz Biotechnology) for 1 week.

Techniques: Control, Two Tailed Test, Fluorescence, Injection, Migration, Derivative Assay

Journal: World Journal of Gastrointestinal Surgery

Article Title: Research progress on O-GlcNAcylation in the occurrence, development, and treatment of colorectal cancer

doi: 10.4240/wjgs.v13.i2.96

Figure Lengend Snippet: Summary of Studies on O-GlcNAcylation and colorectal cancer in recent 5 years

Article Snippet: Harosh-Davidovich et al [ ] , 2018 , CT26 murine colon carcinoma cells and NIH-3T3 murine fibroblasts , ATCC , (1) Protein extraction. β-Catenin IP. (2) Affinity purification of β-catenin with Wheat Germ Agglutinin (WGA). (3) WB. (4) Cell motility assay. (5) OGA and OGT silencing. (6) Luciferase reporter assays. (7) qRT-PCR. And (8) In vivo orthotopic mouse model of CRC , O-GlcNAcylation may enhance the proliferation and metastasis of CRC by regulating the expression of catenin and E-cadherin, which proves the influence of O-GlcNAcylation on the poor prognosis of CRC patients.

Techniques: Glycoproteomics, Quantitative Proteomics, Plasmid Preparation, Transfection, Isolation, Purification, RNA Extraction, Protein Extraction, Western Blot, Flow Cytometry, Biomarker Discovery, Expressing, Migration, Immunofluorescence, Confocal Microscopy, Invasion Assay, Co-culture Assay, Gene Expression, Chromatin Immunoprecipitation, Construct, CRISPR, Knock-Out, Knockdown, shRNA, Marker, Activation Assay, Labeling, Microarray, Immunohistochemistry-IF, Activity Assay, Soft Agar Assay, Ligation, In Vitro, Cell Culture, Stable Transfection, CCK-8 Assay, Virus, Reporter Assay, Reverse Transcription, Viability Assay, Colony Assay, Cell Migration Assay, Infection, Microscopy, Mutagenesis, Immunohistochemical staining, Staining, Sequencing, DNA Methylation Assay, Mass Spectrometry, Multiplex Assay, Immunohistochemistry, Proteomic Assay, Affinity Purification, Motility Assay, Luciferase, In Vivo, Fluorescence, In Situ Hybridization, Sample Prep, Concentration Assay, Chemotaxis Assay, Extraction, Bacteria, Adjuvant, Histopathology, Cell Adhesion Assay, Colorimetric Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intestinal CCL11 and eosinophilic inflammation is regulated by myeloid cell-specific RelA/p65 in mice

doi: 10.4049/jimmunol.1200057

Figure Lengend Snippet: A, Representative flow cytometry plots and histograms of phospho-RelA/p65 expression in F4/80+CD11b+Ly6Chi colonic MΦs from DSS-treated WT (open histogram) and RelA/p65Δmye mice (filled histogram). B. Quantification of Nfkbia expression from Ly6Chi MΦs/monocytes sorted from the peripheral blood or colon of DSS-treated mice. Data represent the mean ± SEM of n = 5 mice per group. Representative western blot analysis of WT (+) and RelA/p65Δmye (−) (C) BMDMs stimulated with 1 ug/mL LPS for 1 hour, (D), colonic epithelium and spleen of total RelA/p65 and/or phospho-RelA/p65. E, representative western blot analysis of IKKα, IκBα, IκBβ, c-Rel, p105 and actin expression in BMDMs stimulated with LPS for indicated times (minutes). F, WT and RelA/p65Δmye BMDMs were stimulated for 24 hours with vehicle or 1 ug/mL LPS and levels of IL-6, TNF-α, IL-1β, NO2 and IL-12p40 were measured in the supernatants. G, Representative flow cytometry plots and graph of BMDMs stimulated for 24 hours with control, IL-4 or IFN-γ + LPS and assessed for active caspase-3. (F and G) Data represent the mean ± SEM of n = 3 individual samples per group. (A, C, E–G) Data is representative of duplicate experiments. Significant differences (*p<0.05**, p < 0.01; ***p < 0.001) between groups.

Article Snippet: The following antibodies were used: rabbit-anti-IKKα, IκBβ, c-Rel, p105/p50 (Santa Cruz; Santa Cruz, CA), phosphoserine 536 RelA/p65, IκBα (Cell Signaling; Danvers MA), and total RelA/p65 (Rockland Immunochemicals, Gilbertsville, Pa) followed by goat anti-rabbit peroxidase-conjugated antibody (Calbiochem; Darmstadt, Germany) and ECL-plus detection reagents (GE Healthcare, Buckinghamshire, United Kingdom).

Techniques: Flow Cytometry, Expressing, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intestinal CCL11 and eosinophilic inflammation is regulated by myeloid cell-specific RelA/p65 in mice

doi: 10.4049/jimmunol.1200057

Figure Lengend Snippet: A, Representative flow cytometry plots and B, quantification of the F4/80+CD11b+Ly6Chi MΦ populations from the colon of WT and RelA/p65Δmye mice following 5 days of vehicle (baseline) or DSS exposure. MΦs were initially gated by SSC vs. FSC followed by F4/80+, CD11b+ and Ly6Chi. C, Representative flow cytometry plots and D, quantification of the F4/80−CD11bhi neutrophil population from the colon of WT and RelA/p65Δmye mice following 5 days of vehicle (baseline) or DSS exposure. MΦs were initially gated by SSC vs. FSC followed by F4/80− and CD11bhi. B and D, data represents the mean ± SEM of n = 5–6 mice per group and is representative of duplicate experiments. Significant differences (*p < 0.05; ***p < 0.001) between groups. n.s. – not significant.

Article Snippet: The following antibodies were used: rabbit-anti-IKKα, IκBβ, c-Rel, p105/p50 (Santa Cruz; Santa Cruz, CA), phosphoserine 536 RelA/p65, IκBα (Cell Signaling; Danvers MA), and total RelA/p65 (Rockland Immunochemicals, Gilbertsville, Pa) followed by goat anti-rabbit peroxidase-conjugated antibody (Calbiochem; Darmstadt, Germany) and ECL-plus detection reagents (GE Healthcare, Buckinghamshire, United Kingdom).

Techniques: Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intestinal CCL11 and eosinophilic inflammation is regulated by myeloid cell-specific RelA/p65 in mice

doi: 10.4049/jimmunol.1200057

Figure Lengend Snippet: A, Representative photomicrographs of anti-MBP-stained colonic sections from baseline and DSS-treated mice (day 6, 2.5%) and colonic eosinophil levels in the colon. B, CCL11 levels in punch biopsy supernatants from WT and RelA/p65Δmye mice in vehicle- (baseline) and DSS-treated mice (day 5, 2.5%) Data indicates the mean ± SEM of n = 6–8 mice per group from triplicate experiments. Significant differences (*p < 0.05; ***p < 0.001) between groups. Magnification of photomicrographs is ×100.

Article Snippet: The following antibodies were used: rabbit-anti-IKKα, IκBβ, c-Rel, p105/p50 (Santa Cruz; Santa Cruz, CA), phosphoserine 536 RelA/p65, IκBα (Cell Signaling; Danvers MA), and total RelA/p65 (Rockland Immunochemicals, Gilbertsville, Pa) followed by goat anti-rabbit peroxidase-conjugated antibody (Calbiochem; Darmstadt, Germany) and ECL-plus detection reagents (GE Healthcare, Buckinghamshire, United Kingdom).

Techniques: Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intestinal CCL11 and eosinophilic inflammation is regulated by myeloid cell-specific RelA/p65 in mice

doi: 10.4049/jimmunol.1200057

Figure Lengend Snippet: A, Representative flow cytometry plot of CD11b+Ly6Chi colonic MΦs flow sorted on day 5 of DSS treatment. B, gene expression was analyzed by qRT-PCR. 3–4 mice were pooled per sample from DSS-treated WT and RelA/p65Δmye mice (3–4 samples per group). Data represents the mean ± SEM. Data is representative of duplicate experiments. Significant differences (*p < 0.05; **p < 0.01) between groups. n.s. – not significant.

Article Snippet: The following antibodies were used: rabbit-anti-IKKα, IκBβ, c-Rel, p105/p50 (Santa Cruz; Santa Cruz, CA), phosphoserine 536 RelA/p65, IκBα (Cell Signaling; Danvers MA), and total RelA/p65 (Rockland Immunochemicals, Gilbertsville, Pa) followed by goat anti-rabbit peroxidase-conjugated antibody (Calbiochem; Darmstadt, Germany) and ECL-plus detection reagents (GE Healthcare, Buckinghamshire, United Kingdom).

Techniques: Flow Cytometry, Expressing, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intestinal CCL11 and eosinophilic inflammation is regulated by myeloid cell-specific RelA/p65 in mice

doi: 10.4049/jimmunol.1200057

Figure Lengend Snippet: A. Heat map showing the clustering of the top 25 differentially-expressed genes following DSS-induced colitis (day 6 vs. day 0, p < 0.05). B. Top 10 differentially-expressed genes following DSS-induced colitis (day 6 vs. day 0, p < 0.05, fold change > 10) that correlated with Ccl11 expression (r > 0.75). C. Pearson product-moment correlation between normalized microarray expression values of Ear5, S100a8 and S100a9 are plotted against Ccl11 from individual animals. (D) Pearson product-moment correlation between S100a8 and S100a9/Hprt ratio and eosinophils/hpf in the colon of WT C57BL6 mice on day 3, 5 and 7 after DSS exposure. E. Representative flow plots of RAGE expression on (E.) colonic F4/80+ CD11b+ Ly6Chi monocytes/MΦ from DSS-treated mice and (F.) BMMΦ. G. CCL11 levels in supernatants from WT and RelA/p65Δmye BMMΦ following 24 hour stimulation with S100a8/S100a9 complex. G. Data represents the mean ± SEM. Data is representative of duplicate experiments. Significant differences (**p < 0.01) between groups.

Article Snippet: The following antibodies were used: rabbit-anti-IKKα, IκBβ, c-Rel, p105/p50 (Santa Cruz; Santa Cruz, CA), phosphoserine 536 RelA/p65, IκBα (Cell Signaling; Danvers MA), and total RelA/p65 (Rockland Immunochemicals, Gilbertsville, Pa) followed by goat anti-rabbit peroxidase-conjugated antibody (Calbiochem; Darmstadt, Germany) and ECL-plus detection reagents (GE Healthcare, Buckinghamshire, United Kingdom).

Techniques: Expressing, Microarray