Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hypoxia-associated circRNA profiling and expression characteristics of Hsa_circ_0000566 in osteosarcoma (OS). (A) CircRNA microarray analysis reveals 35 upregulated and 23 downregulated circRNAs in OS cells under normoxic and hypoxic conditions. The black arrow represents Hsa_circ_0000566. (B) OS cells incubated under various oxygen concentrations. Total RNA extraction was performed for qRT-PCR assay. Western blotting was performed to determine the protein level of HIF-1α. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. Scale bars, 200 μm. (C) Hsa_circ_0000566 expression is much higher in primary OS tissue than in chondroma tissue. Results are representative images according to three different experiments. (D) Quantitative real-time polymerase chain reaction (qRT-PCR) results comparing Hsa_circ_0000566 mRNA expression in 12 OS and chondroma samples. Results are reported as mean ± SD, *p < 0.05, n = 12. (E) Hsa_circ_0000566 expression levels in hFOB1.19 and various OS cell lines. Results are reported as mean ± SD, *p < 0.05, n = 3. (F) Schematic diagram showing Hsa_circ_0000566 back-spliced by exons 2-11 of the VRK1 gene and the corresponding Sanger sequencing. (G) RT-PCR results validating the presence of Hsa_circ_0000566 in 143B and HOS cells. Various primers amplified the Hsa_circ_0000566 region in cDNA but not in genomic DNA. β-actin was used as the negative control. Divergent primers are presented as the opposite direction of the arrowhead, and the convergent primers were shown as the face-to-face direction of the arrowhead. (H) RT-PCR results indicating Hsa_circ_0000566 and VRK1 mRNA expression in untreated 143B and HOS cells and in the cells subjected to treatment with RNase-R. (I) RNA fluorescence in situ hybridization (FISH) results revealing Hsa_circ_0000566 localized mainly in the cytoplasm. Hsa_circ_0000566 probes were labeled with cy3 and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Scale bars, 100 μm. (J) qRT-PCR determination of the main localization of Hsa_circ_0000566 in OS cells. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Microarray, Incubation, RNA Extraction, Quantitative RT-PCR, Western Blot, Standard Deviation, Real-time Polymerase Chain Reaction, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Fluorescence, In Situ Hybridization, Labeling, Staining

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 contributes to in vitro osteosarcoma (OS) cell progression under hypoxic conditions. (A) Hsa_circ_0000566 overexpression and knockdown induced and repressed OS cell proliferation under hypoxia. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. Circ_0000566 represents Hsa_circ_0000566 overexpression, and si circ_0000566 represents Hsa_circ_0000566 knockdown. Vector and Si NC represents the negative control of Hsa_circ_0000566 overexpression and Hsa_circ_0000566 knockdown, respectively. (B) EdU exhibits the impact of Hsa_circ_0000566 on OS cell proliferation under hypoxia. Nuclei are stained with 4’,6-diamidino-2-phenylindole (DAPI). Results are reported as mean ± SD, *p < 0.05, n = 3. Scale bars, 100 μm. (C) Colony formation experiment verifies Hsa_circ_0000566 functions in OS cells under hypoxia. Results are reported as mean ± SD, *p < 0.05, n = 3. (D) Soft agar colony formation assay indicates the effects of Hsa_circ_0000566 on 143B and HOS cell colony forming capacity under hypoxia. Results are reported as mean ± SD, *p < 0.05, n = 3. Scale bars, 100 μm. (E) OS cell migration capacity as determined by Transwell™ migration assays. Results are reported as mean ± SD, *p < 0.05, n = 3. Scale bars, 100 μm. (F) Flow cytometry verifies Hsa_circ_0000566 functions in OS cell apoptosis. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vitro, Over Expression, Knockdown, Standard Deviation, Plasmid Preparation, Negative Control, Staining, Soft Agar Assay, Migration, Flow Cytometry

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 accelerates osteosarcoma (OS) glucose metabolism and regulates hypoxia-enhanced glycolysis. (A) Colors of the media indicate that Hsa_circ_0000566 silencing decreased lactate accumulation under hypoxia. (B-C) Quantitative real-time polymerase chain reaction (qRT-PCR) or western blots evaluating the expression levels of genes involved in glucose metabolism in 143B and HOS cells transfected with Hsa_circ_0000566-overexpressing, Hsa_circ_0000566 (shRNA), or vector plasmids. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. (D) Hsa_circ_0000566 knockdown in OS cells with decreased lactate accumulation, while Hsa_circ_0000566 overexpression has increased lactate accumulation. Results are reported as mean ± SD, *p < 0.05, n = 3. (E) Extracellular acidification rate (ECAR) indicates glycolysis rate. ECAR decreases in response to Hsa_circ_0000566 knockdown and increases in response to Hsa_circ_0000566 overexpression. Oxygen consumption rate (OCR) represented mitochondrial respiratory capacity. OCR is enhanced in response to Hsa_circ_0000566 silencing and reduced in response to Hsa_circ_0000566 overexpression in OS cells. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Transfection, shRNA, Plasmid Preparation, Standard Deviation, Knockdown, Over Expression

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 establishes interactions with HIF-1α and confers protection against ubiquitination-mediating degradation. (A) Effects of Hsa_circ_0000566 knockdown and Hsa_circ_0000566 overexpression on mRNA and protein expression in 143B and HOS cells under hypoxia. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. (B) Western blotting results revealing the impact of bortezomib treatment on the changes occurring at HIF-1α protein level mediated by Hsa_circ_0000566 silencing and vector transfection. (C) Western blotting assessment of the impact of CHX treatment on the variations in HIF-1α protein levels affected by Hsa_circ_0000566 silencing and vectors. Results are reported as mean ± SD, *p < 0.05, n = 3. (D) The western blot illustrates the effects of Hsa_circ_0000566 knockdown in the Hyp564 HIF-1α protein levels in the presence or absence of bortezomib treatment. (E) Immunoprecipitation assessing the HIF-1α ubiquitination levels in Hsa_circ_0000566 silencing and Hsa_circ_0000566 overexpressing osteosarcoma (OS) cells under hypoxia. Culture media were supplemented with bortezomib (250 nM) for 6 h. (F) The combination of Hsa_circ_0000566 with HIF-1α confirmed by radioimmunoprecipitation (RIP). Results are reported as mean ± SD, *p < 0.05, n = 3. (G) Pulldown assay validation of the interaction between Hsa_circ_0000566 and HIF-1α. (H) A RIP assay of HIF-1α regions interacting with Hsa_circ_0000566. Schematic diagram shows HIF-1α protein fragments. Results are reported as mean ± SD, *p < 0.05, n = 3. (I) Interaction profile between Hsa_circ_0000566 and HIF-1α obtained from catRAPID (left). (J) Schematic diagram showing Hsa_circ_0000566 RNA fragments. Combinative regions between Hsa_circ_0000566 and HIF-1α were identified by RIP assay. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Ubiquitin Proteomics, Knockdown, Over Expression, Expressing, Standard Deviation, Western Blot, Plasmid Preparation, Transfection, Immunoprecipitation, Biomarker Discovery

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 promotes osteosarcoma (OS) glucose metabolism and tumorigenesis progression in vivo. (A) 143B cells stably transfected with Hsa_circ_0000566 knockdown, HIF-1α overexpression, or empty vector plasmids. Nude mice were subcutaneously injected with 1 × 10 7 cells that were either stable negative controls or those with Hsa_circ_0000566 knockdown, HIF-1α overexpression, or Hsa_circ_0000566 knockdown. Thirty days after injection, the animals were euthanized, and their tumors dissected and photographed. (B) Tumor weight measurements on the same day the mice were euthanized. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 5. (C) Tumor volumes (ab2/2) were calculated every 6 d from the day after the mice were injected with stable OS cells. (D-E) Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) exhibit the expression levels of the genes involved in glycolysis metabolism. Results are reported as mean ± SD, *p < 0.05, n = 3. (F) Fluorescence in situ hybridization (FISH), hematoxylin and eosin (H&E) staining, and immunohistochemistry (IHC) analysis indicate the OS organization in mice and relative GLUT1, GLUT4, PDK1, PDK4, and LDHA protein levels in tumors from different groups. (G) In situ tumor formation experiment reveals that HIF-1α overexpression recovered Hsa_circ_0000566 knockdown-induced tumor attenuation. Results are reported as mean ± SD, *p < 0.05, n = 4. (H) Micro-computed tomography (CT) indicates the functions of HIF-1α and Hsa_circ_0000566 knockdown in bone loss. (I) H&E staining of lung metastasis. In mice injected in the tail vein with various stable 143B cells, lung metastasis was detected using an in vivo bioluminescence imaging system. Results are reported as mean ± SD, *p < 0.05, n = 5.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vivo, Stable Transfection, Transfection, Knockdown, Over Expression, Plasmid Preparation, Injection, Standard Deviation, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Fluorescence, In Situ Hybridization, Staining, Immunohistochemistry, In Situ, Micro-CT, Imaging

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: ( A ) Representation of the strategy used for transcriptomic analysis in time and space in the dorsal and lateral OB lineages. pCX-GFP plasmid was introduced into neural stem cells (NSCs) residing within the dorsal or lateral V-SVZ and GFP-positive cells were isolated by FACS at different time points after electroporation (Elpo). The mRNA content was analyzed by micro-array . ( B ) Quantification of Vax1 mRNA expression detected by micro-array analysis in dorsal (brown) and lateral (purple) progenies during neurogenesis. ( C–H ) In situ hybridization revealing Vax1 mRNA (in blue) combined with immuno-histochemistry using antibodies detecting (in brown) PAX6 ( C, C’, G ), ASCL1 ( D, D’ ), DLX2 ( E, E’, H ) or KI67 ( F, F’ ) proteins in the V-SVZ ( C–F ) or RMS ( G, H ) at postnatal day 3 ( P3 ). ( C’–F’ ) High magnification of cellular staining in the V-SVZ (area indicated by the yellow bracket in C-F). Arrows ( C’ ): examples of strong PAX6 only positive cell in the dorso-lateral SVZ; blue staining underneath labels cells from a distinct plane. Arrow heads ( E’, F’ ): double positive cells for DLX2 and KI67, respectively. High magnification of the RMS highlights the differential expression of Vax1 and Pax6 along the dorso-ventral axis ( G’,G” ) and the co-localization with Dlx2 ( H’,H” ). ( I ) Schematic representation of gene expression profile in different cell types of the neurogenic sequence. Circular arrow indicates proliferating cells. LV: lateral ventricle, RG: radial glia, TAP: transit amplified precursor, VZ: ventricular zone, SVZ: sub-ventricular zone. D: dorsal, L: lateral, S: septal, V: ventral. Scale bars: 100 µm ( C–F ), 20 µm ( C’–F’ ), 50 µm ( G–H ).

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Plasmid Preparation, Isolation, Electroporation, Microarray, Expressing, In Situ Hybridization, Immunohistochemistry, Staining, Quantitative Proteomics, Gene Expression, Sequencing, Amplification

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: Cells were analyzed 15 days after electroporation of lateral V-SVZ progenitors by Cre mRNA in WT or Vax1cKO mice. Data are shown as means ± SD, dots represent individual animals. WT: n = 12, Vax1cKO: n = 17. Figure 2—figure supplement 2—source data 1. Quantification of tdTomato+ PGC in Vax1 mutant.

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Electroporation, Mutagenesis

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: ( A ) Strategy used to determine the expression of microRNAs in Vax1-overexpressing progenitors. PCAG-Vax1 and pCX-GFP were simultaneously introduced into NSCs by electroporation of the lateral wall of postnatal P1 brains. Lateral V-SVZ was dissected out 2 days after electroporation and GFP+ cells were isolated by flow cytometry (FACS) to perform quantitative RT-PCR analysis. ( B ) Quantification of Vax1 mRNA level by qRT-PCR in control and Vax1OE conditions, normalized to beta-actin and reported in Vax1 condition as relative level to control, validating the overexpression of Vax1 after electroporation. ( C ) Quantification of miR-7 expression in both conditions. Expression level of miR-7 was normalized by invariant expression of microRNA let-7a and reported in Vax1 condition as relative level to control. Experiments in B and C were performed in triplicate, and data were obtained from ( B ) two independent biological replications or ( C ) three technical replications. ( D ) Genome browser images representing the chromosomal portions encoding the three MiR-7 loci (depicted in pink). Mir-7–1 lies within an intronic sequence of the Hnrnpk gene whereas MiR-7–2 and MiR-7b reside within intergenic sequences. Vax1-binding sites found in the upstream regulatory region of the three MiR-7 are represented by red boxes. ( E ) Model of cross-regulatory interaction between Vax1 , miR-7, and Pax6 in the lateral V-SVZ to control the number of dopaminergic neurons generated by the neural stem cells regionalized in this aspect. This model is supported by our present data and previous work where it was shown that miR-7 was required to inhibit PAX6 expression in lateral NSCs to produce the correct number of dopaminergic neurons in the postnatal OB. Here, we propose that Vax1 acts upstream of miR-7 by positively regulating its expression and consequently inhibiting PAX6. However, it is also possible that Vax1 directly represses the expression of Pax6 mRNA (dashed line) by acting on its promoter . Additionally, Vax1 is required to generate Calbindin neurons from the ventral aspect of the lateral V-SVZ. Figure 5—source data 1. Quantification of expression level of Vax1 and miR-7 in V-SVZ cells.

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Expressing, Electroporation, Isolation, Flow Cytometry, Quantitative RT-PCR, Control, Over Expression, Sequencing, Binding Assay, Generated

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet: ( A ) Strategy used to target lateral V-SVZ NSCs with Cre -mRNA. Tomato+ recombined cells were isolated 2 days after electroporation. ( B ) Vax1 mRNA level quantified by RT-PCR was normalized to beta-actin and reported in Vax1cKO condition as relative level to control (WT).

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Isolation, Electroporation, Reverse Transcription Polymerase Chain Reaction, Control

Journal: eLife

Article Title: Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

doi: 10.7554/eLife.58215

Figure Lengend Snippet:

Article Snippet: CRE Recombinase mRNA (130-101-113, a generous gift from S. Wild and A. Bosio, Miltenyi Biotec, Bergisch Gladbach, Germany) and pCX-CRE ( ) were used at a concentration of 0.5 μg/μl.

Techniques: Sequencing, Recombinant, Plasmid Preparation, SYBR Green Assay, Software, Imaging

Journal: Science Progress

Article Title: Role of thrombus-derived exosomal lncRNA LOC101928697 in regulating endothelial function via FUS protein interaction in myocardial infarction

doi: 10.1177/00368504251372111

Figure Lengend Snippet: Analysis of the correlation between FUS protein and myocardial infarction. (a) Enrichment Analysis Bar Plot based on differential gene expression profiles in lncRNA microarray analysis.(b) Detection information about lncRNA LOC101928697 binding to FUS proteins in AnnoLnc2 database. (c) Detection information about lncRNA LOC101928697 binding to FUS protein in RBPDP database. (d) Scores in the RPISeq database on the model of lncRNA LOC101928697 binding to FUS protein. (e-g) Prediction information about lncRNA LOC101928697 binding to FUS protein in catRAPID website, (e) Statistical map information about protein and RNA binding sites, (f) Total scoring information, and (g) Interaction map showing the interaction region between protein and RNA. (h-i) Analyses about bioinformatics techniques based on GSE163772 in the GEO database, where (h) is a statistical map of FUS gene expression in endothelial cells of a mouse model of myocardial infarction, and (i) A scatter plot about the correlation between the level of FUS gene expression and the disease state (control vs. myocardial infarction).

Article Snippet: After extensive washing, the bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot using anti-FUS antibody (Proteintech, Cat No. 11570-1-AP, dilution 1:5000) to detect the enrichment of FUS protein.

Techniques: Gene Expression, Microarray, Binding Assay, RNA Binding Assay, Control

Journal: Science Progress

Article Title: Role of thrombus-derived exosomal lncRNA LOC101928697 in regulating endothelial function via FUS protein interaction in myocardial infarction

doi: 10.1177/00368504251372111

Figure Lengend Snippet: Interaction of exosomal lncRNA LOC101928697 with FUS proteins. (a and b) The western blot detection of FUS protein expression in each group of cells and the statistical graph. (c) Statistical graph of RT-qPCR to detect the expression of FUS at the mRNA level in each group of cells. (d) The fluorescence graph of fluorescence in situ hybridization (FISH) experiment. In which FUS was labeled with green fluorescence, lncRNA LOC101928697 was labeled with red fluorescence, and the nucleus was labeled with blue fluorescence (20×). (e) Western blot detection of FUS protein following RNA pull-down using sense or antisense LOC101928697 transcripts. (f) Quantification of FUS protein enrichment in sense RNA pull-down versus antisense control, based on densitometric analysis. (g-h) Western blot detection of FUS protein expression in each group of cells after knockdown or overexpression of lncRNA LOC101928697 and the statistical graphs. (i) Statistical graph of mRNA level expression of FUS in each group of cells after knockdown or overexpression of lncRNA LOC101928697 by RT-qPCR assay. a p < 0.05 compared to control group. b p < 0.05 compared to exosome group. c p < 0.05 compared to siRNA + exosome group.

Article Snippet: After extensive washing, the bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot using anti-FUS antibody (Proteintech, Cat No. 11570-1-AP, dilution 1:5000) to detect the enrichment of FUS protein.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Fluorescence, In Situ Hybridization, Labeling, Protein Enrichment, Control, Knockdown, Over Expression

Journal: Genes, chromosomes & cancer

Article Title: Novel Mouse Model Recapitulates Genome and Transcriptome Alterations in Human Colorectal Carcinomas

doi: 10.1002/gcc.22426

Figure Lengend Snippet: KEGG-pathway Comparison of Gene Expression Changes in Human colon cancer and Mouse Model. Displayed here are the murine gene expression patterns derived from murine mRNA microarray analysis from the late-transformed colon cell lines, which were compared with human CRC specific genes as listed in the KEGG pathway. We have superimposed the directionality of the gene expressions of the colon-specific mouse genes onto the human genes using the red shading within the boxes to indicate upregulation, and green shading for downregulation. The red boarders correspond to genes upregulated in the human colon cancer pathway; green boarders reveal those that are downregulated. No shading indicates that no change occurred in expression of the colon cancer-specific mouse genes relative to normal uncultured mouse epithelial cells. Two tumor suppressors associated with human colon cancer, Apc and Dcc were both downregulated in the mouse model. Ccn1b, Smad3, and Tgfb1, which are upregulated in human CRC were also upregulated in the mouse. The two major differences between the two species are for the genes Bcatenin and ERK, both of which are upregulated in human colon cancers, but downregulated in the transformed murine colon epithelial cells.

Article Snippet: The microarrays were hybridized in a rotisserie Microarray Oven (Model 777 SciGene, Sunnyvale, CA) at 658C and speed of 10 rpm for 40 hr.

Techniques: Expressing, Derivative Assay, Microarray, Transformation Assay

Journal: OncoTargets and Therapy

Article Title: Tumor heterogeneity in the recurrence of epithelial ovarian cancer demonstrated by polycomb group proteins

doi: 10.2147/ott.s67570

Figure Lengend Snippet: Figure 2 Genetic heterogeneity underlying the heterogeneous expression of polycomb group proteins. Amplification of BMI1 and EZH2 was observed by fluorescence in situ hybridization in recurrent tumor tissues, with intensive expression of proteins detected by immunohistochemistry simultaneously. (A) BMI1 signals in primary tumors. (B) BMI1 signals in recurrent tumors. (C) EZH2 signals in primary tumors. (D) EZH2 signals in recurrent tumors. Note: Bars represent 25 µm.

Article Snippet: Mouse anti-human BMi1 1:200 abcam, cambridge, UK rabbit anti-human ring1 1:250 abcam, cambridge, UK goat anti-human ring2 1:100 abcam, cambridge, UK rabbit anti-human PhF1 1:50 abcam, cambridge, UK goat anti-human Mel18 1:500 santa cruz Biotechnology, ca, Usa goat anti-human Phc1 1:200 santa cruz Biotechnology, ca, Usa goat anti-human cBX2 1:200 santa cruz Biotechnology, ca, Usa rabbit anti-human cBX4 1:200 abcam, cambridge, UK Mouse anti-human rYBP 1:600 abcam, cambridge, UK rabbit anti-human eZh2 1:200 cell signaling, Danvers, Ma, Usa rabbit anti-human eeD 1:200 abcam, cambridge, UK Mouse anti-human sUZ12 1:500 abcam, cambridge, UK MaxVisionTM hrP-Polymer goat anti-mouse/rabbit ihc Kit \ Maixin Biological Technology Development company, Fuzhou, People’s republic of china MaxVisionTM hrP-Polymer rabbit anti-goat ihc Kit \ Maixin Biological Technology Development company, Fuzhou, People’s republic of china Abbreviations: hrP, horseradish peroxidase; ihc, immunohistochemistry.

Techniques: Expressing, Amplification, Fluorescence, In Situ Hybridization, Immunohistochemistry

Journal: OncoTargets and Therapy

Article Title: Tumor heterogeneity in the recurrence of epithelial ovarian cancer demonstrated by polycomb group proteins

doi: 10.2147/ott.s67570

Figure Lengend Snippet: Figure 3 Epigenetic heterogeneity underlying the heterogeneous expression of polycomb group proteins. Notes: (A) miRNA microarray expression profiling of six pairs of primary and recurrent ovarian tumor tissues: heat map shows eight downregulated (fold change .2) miRNAs in recurrent tumors. (B) Luciferase reporter assay: (B1) miR-4261 and miR-298 significantly suppressed the luciferase activity of ZNF207 and ILF3 (P=0.001, P=0.008); (B2) ZNF207 and ILF3 significantly promoted the luciferase activity of EZH2-promoter reporter plasmid (P=0.000, P=0.000). (C) Transfection of miR-4261 (C1 and C3) and miR-298 (C2 and C4) mimics into ovarian cancer cells, A2780 and OVCAR8, confirmed by real-time polymerase chain reaction and fluorescence. (D) Expression of ZNF207, ILF3, and EZH2 in ovarian cancer cells after miR-4261 and miR-298 upregulation: (D1, D2, and D4) a significant reduction of ZNF207 and ILF3 at mRNA and protein levels was observed (for ZNF207, P=0.001 both; for ILF3, P=0.000, P=0.001); (D3 and D4) EZH2 expression was only concomitantly reduced significantly by miR-298 overexpression (P=0.001 both). Abbreviation: miRNA, microRNA.

Article Snippet: Mouse anti-human BMi1 1:200 abcam, cambridge, UK rabbit anti-human ring1 1:250 abcam, cambridge, UK goat anti-human ring2 1:100 abcam, cambridge, UK rabbit anti-human PhF1 1:50 abcam, cambridge, UK goat anti-human Mel18 1:500 santa cruz Biotechnology, ca, Usa goat anti-human Phc1 1:200 santa cruz Biotechnology, ca, Usa goat anti-human cBX2 1:200 santa cruz Biotechnology, ca, Usa rabbit anti-human cBX4 1:200 abcam, cambridge, UK Mouse anti-human rYBP 1:600 abcam, cambridge, UK rabbit anti-human eZh2 1:200 cell signaling, Danvers, Ma, Usa rabbit anti-human eeD 1:200 abcam, cambridge, UK Mouse anti-human sUZ12 1:500 abcam, cambridge, UK MaxVisionTM hrP-Polymer goat anti-mouse/rabbit ihc Kit \ Maixin Biological Technology Development company, Fuzhou, People’s republic of china MaxVisionTM hrP-Polymer rabbit anti-goat ihc Kit \ Maixin Biological Technology Development company, Fuzhou, People’s republic of china Abbreviations: hrP, horseradish peroxidase; ihc, immunohistochemistry.

Techniques: Expressing, Microarray, Luciferase, Reporter Assay, Activity Assay, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Fluorescence, Over Expression

Journal: Translational oncology

Article Title: MicroRNA-506-3p increases the response to PARP inhibitors and cisplatin by targeting EZH2/β-catenin in serous ovarian cancers.

doi: 10.1016/j.tranon.2020.100987

Figure Lengend Snippet: Fig. 1. Targeting of EZH2 by miR-506-3p. (A) Microarray analysis of EZH2 mRNA after overexpression of miR-506-3p for 48 h in SKOV3, OVCA432 and HeyA8 cell lines. EZH2 expression levels in miR-506-3p treatments are normalized to those in miR-ctrl control treatment cells. (B-C) HeyA8 cell lines were transfected with miR-506–3p mimics or miR-ctrl for 48 h; Western blot analysis showed the changes of EZH2 and 𝛽-catenin level. The expression levels of EZH2 and 𝛽-catenin were examined with Image J. (D) TargetScan predicted that the EZH2 3 ′ -UTR has a miR-506-3p binding site, which is highly conserved in different species. (E) Luciferase reporter assay showed that miR-506-3p directly targets the EZH2 3 ′ -UTR. HeLa cells were co-transfected with EZH2 3 ′ -UTR-luciferase reporter, wild-type or mutant, and miR-506-3p mimic or miR-ctrl for 48 h before analysis. Firefly luciferase activity of the reporter was normalized to the internal Renilla luciferase activity. 3 ′ -UTR = 3 ′ -untranslated region. MT = mutant; WT = wild-type.

Article Snippet: The relative mRNA levels of each sample were deterined by the Ct method with the housekeeping gene glyceraldehyde-3hosphate dehydrogenase (GAPDH). estern blot analysis Primary β-actin antibody (rabbit), EZH2 antibody (rabbit) and β- atenin antibody (rabbit) were obtained from Cell Signaling Technology Cell Signaling, USA).

Techniques: Microarray, Over Expression, Expressing, Control, Transfection, Western Blot, Binding Assay, Luciferase, Reporter Assay, Mutagenesis, Activity Assay

Journal: Translational oncology

Article Title: MicroRNA-506-3p increases the response to PARP inhibitors and cisplatin by targeting EZH2/β-catenin in serous ovarian cancers.

doi: 10.1016/j.tranon.2020.100987

Figure Lengend Snippet: Fig. 2. EZH2 and miR-506–3p–induced increases in PARPi/cisplatin sensitivity in ovarian cancer cells. (A,B) HeyA8 cells were transfected with 50 nM si-ctrl or si-EZH2. After 48 h, cells were reseeded for olaparib (A) or cisplatin (B) sensitivity assay. Cell viability was assessed by CCK8 assay. (C–E) HeyA8 cells were co-transfected with EZH2 without the 3 ′ -UTR or empty vector (EV) together with 20 nM miR-506-3p or miR-ctrl. After 24 h, cells were harvested for western blot analysis (C) or reseeded for olaparib (D) or cisplatin (E) sensitivity assay.

Article Snippet: The relative mRNA levels of each sample were deterined by the Ct method with the housekeeping gene glyceraldehyde-3hosphate dehydrogenase (GAPDH). estern blot analysis Primary β-actin antibody (rabbit), EZH2 antibody (rabbit) and β- atenin antibody (rabbit) were obtained from Cell Signaling Technology Cell Signaling, USA).

Techniques: Transfection, Sensitive Assay, CCK-8 Assay, Plasmid Preparation, Western Blot

Journal: Translational oncology

Article Title: MicroRNA-506-3p increases the response to PARP inhibitors and cisplatin by targeting EZH2/β-catenin in serous ovarian cancers.

doi: 10.1016/j.tranon.2020.100987

Figure Lengend Snippet: Fig. 3. EZH2 regulates 𝛽-catenin signal pathway. (A) Two ovarian cancer cell lines were transfected with EZH2 without the 3 ′ -UTR or empty vector for 48 h, Western blot analysis showed the changes of 𝛽-catenin level. (B) Knockdown of EZH2 by si-EZH2-1, si-EZH2-2 or si-EZH2-3 compared with si-ctrl transfection in HeyA8 and SKOV3 cells, 𝛽-catenin expression by Western blot are shown.

Article Snippet: The relative mRNA levels of each sample were deterined by the Ct method with the housekeeping gene glyceraldehyde-3hosphate dehydrogenase (GAPDH). estern blot analysis Primary β-actin antibody (rabbit), EZH2 antibody (rabbit) and β- atenin antibody (rabbit) were obtained from Cell Signaling Technology Cell Signaling, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Knockdown, Expressing

Journal: Translational oncology

Article Title: MicroRNA-506-3p increases the response to PARP inhibitors and cisplatin by targeting EZH2/β-catenin in serous ovarian cancers.

doi: 10.1016/j.tranon.2020.100987

Figure Lengend Snippet: Fig. 7. MiR-506-3p, EZH2 and 𝛽-catenin ex- pression in an orthotopic mouse model of ovar- ian cancer. (A) MiR-506–3p expression in HeyA8-ip1 tu- mors from control and miR-506-3p treated mice was assessed by miRNA in situ hybridiza- tion. (B-D) Samples of HeyA8-ip1 tumors from control and miR-506-3p treated mice were sub- jected to IHC staining for EZH2 and 𝛽-catenin; Expression of EZH2 and 𝛽-catenin protein was calculated as IHC staining scores.

Article Snippet: The relative mRNA levels of each sample were deterined by the Ct method with the housekeeping gene glyceraldehyde-3hosphate dehydrogenase (GAPDH). estern blot analysis Primary β-actin antibody (rabbit), EZH2 antibody (rabbit) and β- atenin antibody (rabbit) were obtained from Cell Signaling Technology Cell Signaling, USA).

Techniques: Expressing, Control, In Situ, Immunohistochemistry

Journal: Translational oncology

Article Title: MicroRNA-506-3p increases the response to PARP inhibitors and cisplatin by targeting EZH2/β-catenin in serous ovarian cancers.

doi: 10.1016/j.tranon.2020.100987

Figure Lengend Snippet: Fig. 8. MiR-506-3p, EZH2 and 𝛽-catenin expression in 92 ovarian cancer patients. (A) MiR-506–3p expression in ovarian tumors from 92 patients was assessed by miRNA in situ hybridization. (B–D) Samples of ovarian tumors from 92 patients were subjected to IHC staining for EZH2 and 𝛽-catenin; Expression of EZH2 and 𝛽-catenin protein was calculated as IHC staining scores.

Article Snippet: The relative mRNA levels of each sample were deterined by the Ct method with the housekeeping gene glyceraldehyde-3hosphate dehydrogenase (GAPDH). estern blot analysis Primary β-actin antibody (rabbit), EZH2 antibody (rabbit) and β- atenin antibody (rabbit) were obtained from Cell Signaling Technology Cell Signaling, USA).

Techniques: Expressing, In Situ Hybridization, Immunohistochemistry

Journal: Oncogene

Article Title: Breast cancer stem cell-derived exosomal lnc-PDGFD induces fibroblast-niche formation and promotes lung metastasis

doi: 10.1038/s41388-024-03237-4

Figure Lengend Snippet: A Biodistribution of PKH26-labeled exosomes (red) in the potential metastatic organs of mice treated with non-BCSCs and BCSCs released exosomes. PBS were used as control. Scale bar, 100 μm. B Quantitative analysis of fluorescence exosomes in the potential metastatic organs ( n = 5 random microscopic field). C Immunofluorescence (IF) analysis the distribution of PKH26-labeled (red) in resident tissue-specific stomal cells in lung. Representative IF images of exosomes co-staining with α-SMA, S100A4, CD31 and F4/80 in lung tissue sections from mice. Scale bar, 25 μm. D Quantitative analysis of fluorescence exosomes in α-SMA + cells, S100A4 + cells, CD31 + cells and F4/80 + cells in lung of mice ( n = 5 random microscopic field). E Representative IF images of α-SMA, S100A4, Vimentin and Fibronectin expression in lung tissue of mice educated with PBS, non-BCSC-Exos and BCSC-Exos for 3 weeks. Scale bar, 100 μm. ( n = 5 random microscopic field). F Western blot analysis of fibroblasts activation associated proteins, α-SMA, FAP, Vimentin and Fibronectin in the lung fibroblasts of mice from each group. G Indicated genes expression in lung fibroblasts cells of mice were detected by qRT-PCR analysis. Data were presented the mean ± SD, p values were calculated using two-tailed Student’s t test for ( G ), two-way ANOVA followed by multiple comparisons for B , and D . * p < 0.05, ** p < 0.01, *** p < 0.001, ns no significance.

Article Snippet: The following primary antibodies were used: Alix (Proteintech, 12422-1-AP, 1:1000 dilution), TSG101 (Proteintech, 28283-1-AP, 1:1000 dilution), CD63 (Proteintech, 25682-1-AP, 1:1000 dilution), CD9 (Proteintech, 20597-1-AP, 1:1000 dilution), HSP70 (Proteintech, 10995-1-AP, 1:1000 dilution), GAPDH (Proteintech, 12422-1-AP, 1:1000 dilution), GAPDH (Cell Signaling, #2118, 1:1000 dilution), α-SMA (Cell Signaling, #19245, 1:1000 dilution), S100A4 (Cell Signaling, #13018, 1:1000 dilution), FAP (Santa Cruz, sc-10528, 1:200 dilution), Fibronectin (Cell Signaling, #26836, 1:1000 dilution), Vimentin (Proteintech, 10366-1-AP, 1:1000 dilution), YBX1 (Abcam, ab12148, 1:1000 dilution), Histon H3 (Cell Signaling, #9715, 1:1000 dilution), p65 (Cell Signaling, #8242, 1:1000 dilution), p-p65 (Cell Signaling, #3033, 1:1000 dilution), MYH9 (Proteintech, 11128-1-AP, 1:1000 dilution), TPM3 (Proteintech, 10737-1-AP, 1:1000 dilution), Nanog (Cell Signaling, #8822, 1:1000 dilution), Sox2 (Cell Signaling, #2748, 1:1000 dilution), Oct-4 (Cell Signaling, #2750, 1:1000 dilution), ALDH1A1 (Abcam, ab52492, 1:1000 dilution), and E-cadherin (Proteintech, 20874, 1:1000 dilution) and slug (Proteintech, 12129, 1:1000 dilution).

Techniques: Labeling, Control, Fluorescence, Immunofluorescence, Staining, Expressing, Western Blot, Activation Assay, Quantitative RT-PCR, Two Tailed Test

Journal: Oncogene

Article Title: Breast cancer stem cell-derived exosomal lnc-PDGFD induces fibroblast-niche formation and promotes lung metastasis

doi: 10.1038/s41388-024-03237-4

Figure Lengend Snippet: A Microarray analysis of lncRNAs from exosomes derived from non-BCSCs and BCSCs. The top 50 upregulated and downregulated lncRNAs in BCSC-Exos were presented in the heatmap. B Upregulation of lncRNAs in BCSCs derived exosomes were verified by qRT-PCR analysis. C Expression of lnc-PDGFD in potential metastatic organs in mice treated with PBS, non-BCSC-Exos and BCSC-Exos. D Phalloidin-labeled (green) MRC-5 and WI-38 cells were co-cultured with PKH26-labeled (red) BCSCs derived exosomes, and the fluorescence signals were detected by confocal microscopy. Scale bar, 5 μm. E Fluorescence in suit hybridization showed the expression of lnc-PDGFD in lung fibroblast cells after incubating with non-BCSC-Exos and BCSC-Exos. Scale bar, 25 μm. F Western blot analysis of the expression of fibroblasts activation associated proteins in MRC-5 and WI-38 cells treated with exosomes derived from lnc-PDGFD-overexpression non-BCSCs or lnc-PDGFD-silent BCSCs relative to their respective control cells. G Quantifying the migration ability of MRC-5 and WI-38 cells treated with indicated exosomes. H Quantifying the contraction ability of MRC-5 and WI-38 cells treated with indicated exosomes. I Representative IF images of α-SMA, S100A4, Vimentin and Fibronectin expression in lung tissue of mice educated with indicated exosomes for 3 weeks. Scale bar, 100 μm. ( n = 5 random microscopic field). Data were presented the mean ± SD, p values were calculated using two-tailed Student’s t test for ( B ), ( G ), and ( H ), two-way ANOVA followed by multiple comparisons for C . * p < 0.05, ** p < 0.01, *** p < 0.001, ns no significance.

Article Snippet: The following primary antibodies were used: Alix (Proteintech, 12422-1-AP, 1:1000 dilution), TSG101 (Proteintech, 28283-1-AP, 1:1000 dilution), CD63 (Proteintech, 25682-1-AP, 1:1000 dilution), CD9 (Proteintech, 20597-1-AP, 1:1000 dilution), HSP70 (Proteintech, 10995-1-AP, 1:1000 dilution), GAPDH (Proteintech, 12422-1-AP, 1:1000 dilution), GAPDH (Cell Signaling, #2118, 1:1000 dilution), α-SMA (Cell Signaling, #19245, 1:1000 dilution), S100A4 (Cell Signaling, #13018, 1:1000 dilution), FAP (Santa Cruz, sc-10528, 1:200 dilution), Fibronectin (Cell Signaling, #26836, 1:1000 dilution), Vimentin (Proteintech, 10366-1-AP, 1:1000 dilution), YBX1 (Abcam, ab12148, 1:1000 dilution), Histon H3 (Cell Signaling, #9715, 1:1000 dilution), p65 (Cell Signaling, #8242, 1:1000 dilution), p-p65 (Cell Signaling, #3033, 1:1000 dilution), MYH9 (Proteintech, 11128-1-AP, 1:1000 dilution), TPM3 (Proteintech, 10737-1-AP, 1:1000 dilution), Nanog (Cell Signaling, #8822, 1:1000 dilution), Sox2 (Cell Signaling, #2748, 1:1000 dilution), Oct-4 (Cell Signaling, #2750, 1:1000 dilution), ALDH1A1 (Abcam, ab52492, 1:1000 dilution), and E-cadherin (Proteintech, 20874, 1:1000 dilution) and slug (Proteintech, 12129, 1:1000 dilution).

Techniques: Microarray, Derivative Assay, Quantitative RT-PCR, Expressing, Labeling, Cell Culture, Fluorescence, Confocal Microscopy, Hybridization, Western Blot, Activation Assay, Over Expression, Control, Migration, Two Tailed Test

Journal: Oncogene

Article Title: Breast cancer stem cell-derived exosomal lnc-PDGFD induces fibroblast-niche formation and promotes lung metastasis

doi: 10.1038/s41388-024-03237-4

Figure Lengend Snippet: A Immunofluorescence staining of α-SMA and S100A4 in MRC-5 cells treated with oeVec, lnc-PDGFD and lnc-PDGFD together with si-YBX1. Scale bar, 100 μm. B , C Western blot and qRT-PCR showed the activation of fibroblasts of MRC-5 and MC-38 cells. D Representative immunohistochemical staining of YBX1, α-SMA, Vimentin and Fibronectin of lung metastatic tumors from indicated mice after 3 weeks of education with lnc-PDGFD enriched exosomes or continuous injection of the in-vivo-optimized RNAi. Scale bar, 50 μm. E Schematic illustration of tumor metastasis mice model (upper). Mice were pre-educated with exosomes derived from lnc-PDGFD-overexpression non-BCSCs or treated with in-vivo-optimized si-YBX1 through intravenous injection for 3 weeks. Then luciferase labeled MDA-MB-231 cells were used to perform experimental metastasis model by intravenous injection. For each group, 5 female mice were used for quantification. Representative Bioluminescent imaging, quantification of fluorescence intensity ( F ), H&E staining lung sections ( G ) and quantification of lung metastatic foci H . Scale bar, 1 mm. Data were presented the mean ± SD, p values were calculated using two-tailed Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns no significance.

Article Snippet: The following primary antibodies were used: Alix (Proteintech, 12422-1-AP, 1:1000 dilution), TSG101 (Proteintech, 28283-1-AP, 1:1000 dilution), CD63 (Proteintech, 25682-1-AP, 1:1000 dilution), CD9 (Proteintech, 20597-1-AP, 1:1000 dilution), HSP70 (Proteintech, 10995-1-AP, 1:1000 dilution), GAPDH (Proteintech, 12422-1-AP, 1:1000 dilution), GAPDH (Cell Signaling, #2118, 1:1000 dilution), α-SMA (Cell Signaling, #19245, 1:1000 dilution), S100A4 (Cell Signaling, #13018, 1:1000 dilution), FAP (Santa Cruz, sc-10528, 1:200 dilution), Fibronectin (Cell Signaling, #26836, 1:1000 dilution), Vimentin (Proteintech, 10366-1-AP, 1:1000 dilution), YBX1 (Abcam, ab12148, 1:1000 dilution), Histon H3 (Cell Signaling, #9715, 1:1000 dilution), p65 (Cell Signaling, #8242, 1:1000 dilution), p-p65 (Cell Signaling, #3033, 1:1000 dilution), MYH9 (Proteintech, 11128-1-AP, 1:1000 dilution), TPM3 (Proteintech, 10737-1-AP, 1:1000 dilution), Nanog (Cell Signaling, #8822, 1:1000 dilution), Sox2 (Cell Signaling, #2748, 1:1000 dilution), Oct-4 (Cell Signaling, #2750, 1:1000 dilution), ALDH1A1 (Abcam, ab52492, 1:1000 dilution), and E-cadherin (Proteintech, 20874, 1:1000 dilution) and slug (Proteintech, 12129, 1:1000 dilution).

Techniques: Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR, Activation Assay, Immunohistochemical staining, Injection, In Vivo, Derivative Assay, Over Expression, Luciferase, Labeling, Imaging, Fluorescence, Two Tailed Test

Journal: Oncogene

Article Title: Breast cancer stem cell-derived exosomal lnc-PDGFD induces fibroblast-niche formation and promotes lung metastasis

doi: 10.1038/s41388-024-03237-4

Figure Lengend Snippet: A Colony formation ability of MDA-MB-231 cells treated with indicated CM from lnc-PDGFD-overexpressing and knock-out MRC-5 cells, or control cells. B Protein levels of pluripotent transcription factors in MDA-MB-231 cells treated with indicated CM. C Migration and invasion of MDA-MB-231 cells treated with indicated CM. D Representative Bioluminescent imaging of tumor metastasis model. Luciferase labeled MDA-MB-231 cells treated with indicated CM from lnc-PDGFD-overexpressing and knock-out MRC-5 cells, or control cells were intravenous injected into mice. For each group, 5 female mice were used for quantification. E Representative H&E-stained sections of lung and immunohistochemical staining of Ki-67, Nanog and Sox2 from lung metastatic foci. Scale bar, 1 mm. Scale bar, 100 μm. F RT 2 profiler PCR array showed the expression of cytokines in lnc-PDGFD-overexpressing MRC-5 cells and control cells. G ELISA assay of IL-11 secretion from serum of indicated mice in Figure B. H Correction between IL-11 and α-SMA expression in human cardiac fibroblasts (GSE97358). I lnc-PDGFD-overexpressing MRC-5 cells were treated with IL-11 neutralizing antibody or IgG, and the CM were co-cultured with MDA-MB-231 cells. IL-11Rα knock-out and control MDA-MB-231 cells treated with indicated CM from lnc-PDGFD-overexpressing cells. Transwell assays were used to examine the migration and invasion abilities. J Representative Bioluminescent imaging of tumor metastasis model. Luciferase labeled MDA-MB-231 cells were treated with indicated CM and intravenous injected in mice. For each group, 5 female mice were used for quantification. K Representative H&E-stained sections of lung and immunohistochemical staining of Ki-67, Nanog and Sox2 from lung metastatic foci. Scale bar, 1 mm. Scale bar, 100 μm. Data were presented the mean ± SD, p values were calculated using two-tailed Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns no significance.

Article Snippet: The following primary antibodies were used: Alix (Proteintech, 12422-1-AP, 1:1000 dilution), TSG101 (Proteintech, 28283-1-AP, 1:1000 dilution), CD63 (Proteintech, 25682-1-AP, 1:1000 dilution), CD9 (Proteintech, 20597-1-AP, 1:1000 dilution), HSP70 (Proteintech, 10995-1-AP, 1:1000 dilution), GAPDH (Proteintech, 12422-1-AP, 1:1000 dilution), GAPDH (Cell Signaling, #2118, 1:1000 dilution), α-SMA (Cell Signaling, #19245, 1:1000 dilution), S100A4 (Cell Signaling, #13018, 1:1000 dilution), FAP (Santa Cruz, sc-10528, 1:200 dilution), Fibronectin (Cell Signaling, #26836, 1:1000 dilution), Vimentin (Proteintech, 10366-1-AP, 1:1000 dilution), YBX1 (Abcam, ab12148, 1:1000 dilution), Histon H3 (Cell Signaling, #9715, 1:1000 dilution), p65 (Cell Signaling, #8242, 1:1000 dilution), p-p65 (Cell Signaling, #3033, 1:1000 dilution), MYH9 (Proteintech, 11128-1-AP, 1:1000 dilution), TPM3 (Proteintech, 10737-1-AP, 1:1000 dilution), Nanog (Cell Signaling, #8822, 1:1000 dilution), Sox2 (Cell Signaling, #2748, 1:1000 dilution), Oct-4 (Cell Signaling, #2750, 1:1000 dilution), ALDH1A1 (Abcam, ab52492, 1:1000 dilution), and E-cadherin (Proteintech, 20874, 1:1000 dilution) and slug (Proteintech, 12129, 1:1000 dilution).

Techniques: Knock-Out, Control, Migration, Imaging, Luciferase, Labeling, Injection, Staining, Immunohistochemical staining, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Two Tailed Test

Journal:

Article Title: A microarray-based high throughput molecular marker genotyping method: the tagged microarray marker (TAM) approach

doi: 10.1093/nar/gng113

Figure Lengend Snippet: The tagged microarray approach. (a) Two alleles are distinguished by PCR using three primers. The biotinylated PCR products are arrayed onto a solid support and hybridised with detector probes, which recognise tags specific to the two products. (b) Structure of a tag primer. The allele-specific region is separated from the tag by a C18 linker. (c) Tag detector probe. Two partially complementary oligonucleotides carry a tag sequence (the A tag in this case), its reverse complement (A′), a region of cross-homology (B and B′ for this pair) and a fluorochrome each.

Article Snippet: Slides were hybridised in a Grant microarray hybridisation chamber (In Slide Out hybridisation oven; Boekel Scientific, Feasterville, PA) at 100% relative humidity at 37°C for 30 min.

Techniques: Microarray, Sequencing

Journal:

Article Title: A microarray-based high throughput molecular marker genotyping method: the tagged microarray marker (TAM) approach

doi: 10.1093/nar/gng113

Figure Lengend Snippet: The tagged microarray approach applied to a pea genomic retrotransposon insertion. (a) The two alleles differ by the presence or absence of a retrotransposon insertion (PDR1 here). Different PCR products are produced from the two alleles and these can be recognised either by gel electrophoresis or the tagged microarray approach (Fig. ​(Fig.1).1). (b) Microarray image from 384 pea samples assayed for the 1794-1 allele. Each sample was spotted four times in a row. Cy3 fluorescing spots (green) represent the occupied site allele and Cy5 labelled spots (red) show the unoccupied site allele. Arrowed samples were analysed by gel electrophoresis (Fig. ​(Fig.1c).1c). (c) Agarose gel of samples arrowed in (b), together with corresponding fluorescent images taken from the array image. (d) Quantitation of array spot intensities shown in (c). SD, standard deviation.

Article Snippet: Slides were hybridised in a Grant microarray hybridisation chamber (In Slide Out hybridisation oven; Boekel Scientific, Feasterville, PA) at 100% relative humidity at 37°C for 30 min.

Techniques: Microarray, Produced, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, Quantitation Assay, Standard Deviation

Journal:

Article Title: A microarray-based high throughput molecular marker genotyping method: the tagged microarray marker (TAM) approach

doi: 10.1093/nar/gng113

Figure Lengend Snippet: Modifications to the tagged microarray approach. (a) Using tag detectors labelled with different fluorochromes produces different fluorescent outputs from the same samples. Sample JI 2698 contains an occupied 1794-1 allele and sample Therèse is unoccupied. Samples were spotted in triplicate. (b) Using four tag detectors to analyse the allelic state for two loci in the same reaction. Sample JI 2698 is occupied for 1794-1 and unoccupied for UniTpv, sample Therèse is unoccupied for 1794-1 and occupied for UniTpv and sample JI 2055 is unoccupied for both loci. Tag detectors used for both loci produce Cy3 signal (green) from an occupied allele and Cy5 signal (red) from an unoccupied allele.

Article Snippet: Slides were hybridised in a Grant microarray hybridisation chamber (In Slide Out hybridisation oven; Boekel Scientific, Feasterville, PA) at 100% relative humidity at 37°C for 30 min.

Techniques: Microarray

Journal:

Article Title: A microarray-based high throughput molecular marker genotyping method: the tagged microarray marker (TAM) approach

doi: 10.1093/nar/gng113

Figure Lengend Snippet: Microarray image for 1536 pea DNA samples assayed for the 1794-1 allele under the same conditions as Figure ​Figure2b.2b. Red (Cy5) spots indicate samples containing an unoccupied (–) allele, green indicates occupied (+) and yellow indicates both alleles (+/–). Each PCR was spotted once per array.

Article Snippet: Slides were hybridised in a Grant microarray hybridisation chamber (In Slide Out hybridisation oven; Boekel Scientific, Feasterville, PA) at 100% relative humidity at 37°C for 30 min.

Techniques: Microarray

Journal:

Article Title: A microarray-based high throughput molecular marker genotyping method: the tagged microarray marker (TAM) approach

doi: 10.1093/nar/gng113

Figure Lengend Snippet: Detection of barley SNP polymorphisms using the tagged microarray approach. Cultivars Steptoe and Morex have contrasting alleles for SNP 206 and SNP 957. Cy5 fluorescence indicates the presence of one allele and Cy3 the other. Samples were spotted in triplicate. The Cy3 (green) and Cy5 (red) colour-separated images and the two-colour (overlay) image are shown. Quantification of averaged array intensities (in arbitrary fluorescence units) and the Cy3/Cy5 ratios of signal intensities are also shown. SD, standard deviation.

Article Snippet: Slides were hybridised in a Grant microarray hybridisation chamber (In Slide Out hybridisation oven; Boekel Scientific, Feasterville, PA) at 100% relative humidity at 37°C for 30 min.

Techniques: Microarray, Fluorescence, Standard Deviation

Journal:

Article Title: A microarray-based high throughput molecular marker genotyping method: the tagged microarray marker (TAM) approach

doi: 10.1093/nar/gng113

Figure Lengend Snippet: Microarray image for 384 barley DNA samples assayed for the SNP 206 allele. Reaction and detection conditions were the same as for Figure ​Figure55 and arraying format is the same as for Figure ​Figure2b.2b. Each PCR was spotted four times per array.

Article Snippet: Slides were hybridised in a Grant microarray hybridisation chamber (In Slide Out hybridisation oven; Boekel Scientific, Feasterville, PA) at 100% relative humidity at 37°C for 30 min.

Techniques: Microarray

Journal: Cancers

Article Title: Tribbles Pseudokinases in Colorectal Cancer

doi: 10.3390/cancers13112825

Figure Lengend Snippet: Tribbles amplification and overexpression in colon cancer tissues. Main result obtained for each study included in the section is shown for each Tribble member. Details of the samples, databases and methodology used by the authors is detailed under respective column. Whenever available, the number of samples analyzed is specified under n . nd, not defined.

Article Snippet: , Genomic amplification in 7 out of the 15 cell lines tested; 11–14.4% gain in CRC samples. , DLD-1, HCT116, HCT116p53-/-, SW48, LoVo, SW480, SW837, HT29, T84, Colo 201, Colo 320DM, LS411N, SK-CO-1, NCI H508 and NCI H716 cells (ECACC or ATCC); primary CRC samples ( n = 881); CRC tumors ( n = 76). , nd , Oncomine database; Comparative genomic hybridization (CGH) NimbleGen microarray (Roche): GSE72296; Illumina Whole Genome Gene Expression Profiling: GSE72544; Fluorescence in situ hybridisation (FISH): TRIB1 /CEN8p probe (Abnova); Tissue microarray (TMA), automated quantitative analysis (AQUA) with anti- TRIB1 rabbit polyclonal antibody. , Briffa, 2015 [ ] .

Techniques: Amplification, Over Expression, Control, Isolation, Western Blot, Hybridization, Microarray, Gene Expression, Fluorescence, In Situ, Immunohistochemistry, Northern Blot, Real-time Polymerase Chain Reaction, In Situ Hybridization, Quantitative RT-PCR, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Astrocytes upregulate survival genes in tumor cells and induce protection from chemotherapy.

doi: 10.1593/neo.11112

Figure Lengend Snippet: Figure 4. (A) Measured expression patterns of human and mouse genes by cross-species hybridization with human and mouse RNA microarray slides. The data are presented in matrix format in which rows represent individual gene and columns represent each hybrid- ization event. Each cell in the matrix represents the expression level of a gene feature in an individual hybridization. The red in cells reflects measured expression levels, and intensity of color represents the magnitude of expression (log2-transformed scale). These re- sults confirm the specificity of the analyses. (B) Expression patterns of human genes in MDA-MB-231 and PC14Br4 cocultured with murine astrocytes or 3T3 cells. We identified genes whose expression patterns were altered on interaction with astrocytes in each cell line by applying two-sample t tests (P < .001). In MDA-MB-231 and PC14Br4 cells, 1069 and 594 genes, respectively, were differentially expressed. The expression of 205 genes was altered in both cell lines. Of these, several are related to antiapoptosis and cell survival. (C) Validation of microarray by Western blot analysis. Protein was extracted from MDA-MB-231 and PC14Br4 cells after they were cultured alone or with or murine astrocytes. The expression of the antiapoptotic survival genes BCL2L1, TWIST1, and GSTA was upregulated in MDA-MB-231 and PC14Br4 cells cocultured with murine astrocytes. (D) Expression of BCL2L1, TWIST1, and GSTA5 in clinical specimen of breast cancer and lung cancer metastasis in the brain and breast cancer in the brain and lung. BCL2L1, TWIST1, and GSTA5 were highly expressed (green) on tumor cells. Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Tumor cells in clinical specimen of breast cancer metastases to the lung were negative for expression of these genes, whereas low expression of these genes was detected in normal alveolar epithelial cells and alveolar macrophages.

Article Snippet: Slides were hybridized with rabbit polyclonal antibody against BCL2L1 (1:200; Cell Signaling), rabbit polyclonal antibody against TWIST1 (1:200; Cell Signaling), or mouse monoclonal antibody against GSTA5 (1:200; Novus Biologicals).

Techniques: Expressing, Hybridization, Microarray, Transformation Assay, Biomarker Discovery, Western Blot, Cell Culture, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Astrocytes upregulate survival genes in tumor cells and induce protection from chemotherapy.

doi: 10.1593/neo.11112

Figure Lengend Snippet: Figure 5. Chemoprotection requires constant contact with astrocytes. (A) The apoptotic index of PC14Br4 cells cultured with astrocytes was 30.9% ± 3.3%, and with fibroblasts, it was 54.6% ± 0.6% (P > .01). Tumor cells initially cultured with astrocytes were harvested and reincubated with astrocytes or fibroblasts in the presence of taxol for another 72 hours. Tumor cells cocultured again with astro- cytes maintained the relative resistance to taxol compared with tumor cells cocultured (second cycle) with fibroblasts. Tumor cells initially cultured with fibroblasts were not resistant to taxol, but if these tumor cells were then reincubated (second cycle) with astro- cytes, they developed resistance compared with tumor cells cultured again with fibroblasts. (B) Gene expression of BCL2L1, GSTA5, and TWIST1 was determined in PC14Br4 cells cultured with astrocytes or fibroblasts. Similar to data shown in Figure 3, the survival genes were highly expressed in tumor cells cocultured with astrocytes but not with fibroblasts (first cycle). We harvested surviving tumor cells and cocultured them for the second cycle with either astrocytes or fibroblasts. Once again, only tumor cells cocultured with astrocytes (second cycle) expressed higher levels of BCL2L1, GSTA5, and TWIST1.

Article Snippet: Slides were hybridized with rabbit polyclonal antibody against BCL2L1 (1:200; Cell Signaling), rabbit polyclonal antibody against TWIST1 (1:200; Cell Signaling), or mouse monoclonal antibody against GSTA5 (1:200; Novus Biologicals).

Techniques: Cell Culture, Gene Expression

Journal: Neoplasia (New York, N.Y.)

Article Title: Astrocytes upregulate survival genes in tumor cells and induce protection from chemotherapy.

doi: 10.1593/neo.11112

Figure Lengend Snippet: Figure 6. (A) Western blot analysis for validation of knockdown of target genes using siRNA. The results demonstrate specificity of the transfection with siRNA in the down-regulation of corresponding protein expression level. (B) Upper panel. Chemoprotection assay. Reversal of the protection by astrocytes was achieved only when all three genes were knocked down. Lower panel. Transfection of MDA-MD-231 human breast cancer cells with siRNA targeting BCL2L1, GSTA5, and TWIST1 did not affect the apoptosis index. (C) Stable expression of BCL2L1, GSTA5, and TWIST1 genes. Western blot analysis using anti-Myc antibody shows stable overexpression of tar- geted genes at the indicated time points. (D) Effects of overexpression of BCL2L1, GSTA5, and TWIST1 genes of tumor cells on che- moprotection mediated by murine astrocytes. Overexpression of BCL2L2, GSTA5, TWIST1 alone or all three genes increased tumor cell resistance to taxol in the absence of astrocytes (upper panel). Overexpression of single gene or pool of genes did not affect the apop- tosis index of tumor cells in control cultures (lower panel). *P < .01.

Article Snippet: Slides were hybridized with rabbit polyclonal antibody against BCL2L1 (1:200; Cell Signaling), rabbit polyclonal antibody against TWIST1 (1:200; Cell Signaling), or mouse monoclonal antibody against GSTA5 (1:200; Novus Biologicals).

Techniques: Western Blot, Biomarker Discovery, Knockdown, Transfection, Expressing, Over Expression, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Astrocytes upregulate survival genes in tumor cells and induce protection from chemotherapy.

doi: 10.1593/neo.11112

Figure Lengend Snippet: Figure 7. (A) Western blot analysis for expression of AKT/pAKT and MAPK/pMAPK in human breast cancer cells and lung cancer cells cultured alone or cocultured with astrocytes or fibroblasts. Expression of AKT and MAPK was not altered whether tumor cells were cultured alone or cocultured with astrocytes or fibroblasts. However, the expression of phosphorylated AKT or MAPK was up- regulated only in tumor cells cocultured with astrocytes. (B) Deter- mination of the role of BCL2L1, GSTA5, and TWIST1 genes in AKT and MAPK activation. Transfection with nonspecific (NS-siRNA) and combined siRNA (Mixed siRNA) did not affect activation of AKT and MAPK pathways. (C) Determination of the role of AKT and MAPK activation in the regulation of BCL2L1, GSTA5, and TWIST1 gene expression. Inhibition of activation of the AKT and MAPK pathways inhibited the up-regulation of the expression of BCL2L1, GSTA5 and TWIST1 genes. All figures are representative of one experiment of three independent experiments.

Article Snippet: Slides were hybridized with rabbit polyclonal antibody against BCL2L1 (1:200; Cell Signaling), rabbit polyclonal antibody against TWIST1 (1:200; Cell Signaling), or mouse monoclonal antibody against GSTA5 (1:200; Novus Biologicals).

Techniques: Western Blot, Expressing, Cell Culture, Activation Assay, Transfection, Gene Expression, Inhibition

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Insulin-like growth factor binding protein-7 (IGFBP7) functions as a potential tumor suppressor in hepatocellular carcinoma (HCC)

doi: 10.1158/1078-0432.CCR-10-2774

Figure Lengend Snippet: IGFBP7 is downregulated in human HCC samples. A. Analysis of IGFBP7 expression in tissue microarray by immunohistochemistry. C. Fluorescence In Situ Hybridization (FISH) was performed on human HCC samples for IGFBP7 and CEP4 (probe targeting pericentromeric region of chromosome 4). Red: IGFBP7; green: CEP4.

Article Snippet: Plasmids, cell lines, culture condition, viability assays and chemical reagents IGFBP7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001553","term_id":"359465606","term_text":"NM_001553"}} NM_001553 ) human cDNA clone was obtained from Origene Technologies, Inc (Rockville, MD) and cloned into pcDNA3.1(+)-zeo plasmid (Invitrogen).

Techniques: Expressing, Microarray, Immunohistochemistry, Fluorescence, In Situ Hybridization

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Insulin-like growth factor binding protein-7 (IGFBP7) functions as a potential tumor suppressor in hepatocellular carcinoma (HCC)

doi: 10.1158/1078-0432.CCR-10-2774

Figure Lengend Snippet: IGFBP7 is downregulated in human HCC cell lines and by AEG-1. A. Determination of IGFBP7 mRNA expression by real-time PCR in the indicated cells. THLE-3 is normal immortal human hepatocytes. GAPDH was used as normalization control. B. Secreted IGFBP7 protein level in the conditioned media of the indicated cells determined by ELISA. C. Determination of IGFBP7 mRNA expression by real-time PCR in Hep-pc-4 (pc-4) cells and three independent clones of HepG3 cells overexpressing AEG-1. D. Immunofluorescence detection of IGFBP7 protein in Hep-pc-4 (pc-4) and Hep-AEG-1-14 (AEG1-14) cells. E. Immunohistochemical analysis of AEG-1 and IGFBP7 expression in normal liver and matched HCC from the same patient. The figure represents data from one patient. Similar finding was observed in 13 out of 18 HCC patients. For A-C, data represents mean ± SEM of three independent experiments. *: p<0.05.

Article Snippet: Plasmids, cell lines, culture condition, viability assays and chemical reagents IGFBP7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001553","term_id":"359465606","term_text":"NM_001553"}} NM_001553 ) human cDNA clone was obtained from Origene Technologies, Inc (Rockville, MD) and cloned into pcDNA3.1(+)-zeo plasmid (Invitrogen).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Clone Assay, Immunofluorescence, Immunohistochemical staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Insulin-like growth factor binding protein-7 (IGFBP7) functions as a potential tumor suppressor in hepatocellular carcinoma (HCC)

doi: 10.1158/1078-0432.CCR-10-2774

Figure Lengend Snippet: Overexpression of IGFBP7 inhibits growth of AEG-1-overexpressing cells. Stable clones of Hep-AEG1-14 cells expressing IGFBP7 (IGFBP7-11 and IGFBP7-12) were generated by selection with zeocin. Zeocin-resistant clone of Hep-AEG1-14 cells (Control-2) served as a control. A. IGFBP7 mRNA expression in the indicated cells detected by real-time PCR. B. Secreted IGFBP7 protein level in the indicated cells detected by ELISA. C. Cell viability (MTT) assay of the indicated cells. D. Colony formation assay of the indicated cells. For A-D, data represents mean ± SEM of three independent experiments. *: p<0.05. E. Western blot analysis performed in the indicated cells with the indicated antibodies. EF1α was used as loading control.

Article Snippet: Plasmids, cell lines, culture condition, viability assays and chemical reagents IGFBP7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001553","term_id":"359465606","term_text":"NM_001553"}} NM_001553 ) human cDNA clone was obtained from Origene Technologies, Inc (Rockville, MD) and cloned into pcDNA3.1(+)-zeo plasmid (Invitrogen).

Techniques: Over Expression, Clone Assay, Expressing, Generated, Selection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, MTT Assay, Colony Assay, Western Blot

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Insulin-like growth factor binding protein-7 (IGFBP7) functions as a potential tumor suppressor in hepatocellular carcinoma (HCC)

doi: 10.1158/1078-0432.CCR-10-2774

Figure Lengend Snippet: IGFBP7 induces senescence. A. Photomicrograph of Control-2, IGFBP7-11 and IGFBP7-12 clones of Hep-AEG-1-14 cells stained for senescence-associated β-galactosidase (SA-β-gal) after 1 week of culture. B. Graphical representation of quantification of SA-β-gal positive cells. At least 1,000 cells were counted for each group. Data represents mean ± SEM of three independent experiments. *: p<0.05. C. Photomicrograph of Control-2, IGFBP7-11 and IGFBP7-12 clones of Hep-AEG-1-14 cells stained for γ-H2AX and counterstained with DAPI to stain the nucleus. D. B. Graphical representation of quantification of γ-H2AX foci/cell. At least 100 cells were scored for each group. Data represents mean ± SEM of three independent experiments. *: p<0.05.

Article Snippet: Plasmids, cell lines, culture condition, viability assays and chemical reagents IGFBP7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001553","term_id":"359465606","term_text":"NM_001553"}} NM_001553 ) human cDNA clone was obtained from Origene Technologies, Inc (Rockville, MD) and cloned into pcDNA3.1(+)-zeo plasmid (Invitrogen).

Techniques: Clone Assay, Staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Insulin-like growth factor binding protein-7 (IGFBP7) functions as a potential tumor suppressor in hepatocellular carcinoma (HCC)

doi: 10.1158/1078-0432.CCR-10-2774

Figure Lengend Snippet: Overexpression of IGFBP7 inhibits AEG-1-mediated tumorigenesis in nude mice. Subcutaneous xenografts were established in athymic nude mice using Control-2, IGFBP7-11 and IGFBP7-12 clones of Hep-AEG-1-14 cells. A. A representative photograph of tumor-bearing mice at the end of the study. B. Measurement of tumor volume at the indicate time point. Data represents mean ± SEM. *: p<0.05. C. Tumor sections were immunostained for IGFBP7, CD31 and Ki-67.

Article Snippet: Plasmids, cell lines, culture condition, viability assays and chemical reagents IGFBP7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001553","term_id":"359465606","term_text":"NM_001553"}} NM_001553 ) human cDNA clone was obtained from Origene Technologies, Inc (Rockville, MD) and cloned into pcDNA3.1(+)-zeo plasmid (Invitrogen).

Techniques: Over Expression, Clone Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Insulin-like growth factor binding protein-7 (IGFBP7) functions as a potential tumor suppressor in hepatocellular carcinoma (HCC)

doi: 10.1158/1078-0432.CCR-10-2774

Figure Lengend Snippet: IGFBP7 inhibits angiogenesis. A. Control-2, IGFBP7-11 and IGFBP7-12 clones of Hep-AEG1-14 cells were implanted in chicken chorioallantoic membrane (CAM) and neovascularization was photographed. B. Graphical representation of new blood vessel formation in CAM when the indicated cells were implanted. The numbers indicate percentage of new blood vessels arising from the existing blood vessels in naïve CAM when VEGF-treated CAM was considered as 100%. Data represents mean ± SEM. *: p<0.05. C. HUVECs were treated with conditioned media from the indicated cells and tube formation was photographed. D. Graphical representation of tube formation by HUVEC treated with conditioned media from the indicated cells. The numbers indicate percentage of tube-like structures when VEGF-treated tube formation was considered as 100%. The data represents mean ± SEM. *: p<0.05.

Article Snippet: Plasmids, cell lines, culture condition, viability assays and chemical reagents IGFBP7 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001553","term_id":"359465606","term_text":"NM_001553"}} NM_001553 ) human cDNA clone was obtained from Origene Technologies, Inc (Rockville, MD) and cloned into pcDNA3.1(+)-zeo plasmid (Invitrogen).

Techniques: Clone Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

doi: 10.1002/stem.1070

Figure Lengend Snippet: The Cnot genes maintain self-renewal by repressing early trophectoderm (TE) transcription factors. (A): Cnot1, Cnot2, and Cnot3 knockdown did not immediately affect known self-renewal factors and pathways. Oct4GiP cells were transfected with control-siRNA (Control), Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), or Cnot3-siRNA2 (Cnot3-KD) in M15 medium. Cells were collected 48 hours after transfection, and total Stat3, Smad1, b-Catenin as well as phospho-Stat3, phospho-Smad1, phosphor-b-Catenin, Oct4, and Nanog levels were determined by Western blot. Starved: control-transfected ESCs cultured in serum-free and LIF-free medium for additional 4 hours. (B): Comparing gene expression changes caused by perturbations of known self-renewal factors: Cnot1, 2, and 3 silencing induced similar changes to those of Oct4 or Sox2 silencing. Pearson's correlation coefficients were calculated between microarray datasets and depicted in a heatmap. The self-renewal factors were clustered by unsupervised hierarchical clustering based on the correlation coefficients. Microarray datasets used for this plot are listed in Supporting Information Table 2. (C): Cnot2 or Cnot3 overexpression cannot rescue Oct4 or Sox2 silencing-induced differentiation. Oct4GiP cells and Oct4GiP cells overexpressing Cnot2 (Cnot2-Rescue, same as in Fig. 1C) or Cnot3 (Cnot3-Rescue, same as in Fig. 1C) were transfected with control, Oct4 (Oct4-KD), or Sox2 (Sox2-KD) siRNAs, and the % differentiation was determined by the Oct4GiP reporter assay. (D): Cnot1, Cnot2, and Cnot3 knockdown induced TE differentiation in the presence of sustained Oct4 expression. ZHBTc4 cells that constitu-tively express Oct4 at the normal level from a Tet-Off promoter were transfected with control or Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), Cnot3-siRNA2 (Cnot3-KD), and the expression of TE markers Cdx2 and Gata3 was determined by qRT-PCR after 4 days. (E): Cdx2 deletion partially rescued Cnot1, Cnot2, and Cnot3 silencing-induced differentiation. Oct4GiP (WT) or dKO23-5 (Cdx2-/- ) cells were transfected with Control-siRNA (Control), Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), or Cnot3-siRNA2 (Cnot3-KD), and the expression of lineage markers was determined by qRT-PCR 96-hour after transfection. Abbreviations: ESC, embryonic stem cell; KD, Knockdown; WT, wild type.

Article Snippet: Primary antibodies used in this study were as follows Oct4 (Abcam, Cambridge, MA; Cat#AB19857, www.abcam.com ), Nanog (Millipore, Billerica, MA; Cat#AB9220, www.millipore.com ), Smad1-5-8-phospho-specific (Cell Signaling Technology, Beverly, MA; Cat#9512, www.cellsignal.com ), Smad1 (Cell Signaling Technology, Cat#9511), Stat3-S727-phosphospecific (Cell Signaling Technology, Cat#9134), Stat3Y705-phospho-specific (Cell Signaling Technology, Cat#9145), Stat3 (Cell Signaling Technology, Cat#9132), b-Catenin-Ser45-phospho-specific (Cell Signaling Technology, Cat#9564), b-Catenin (Cell Signaling Technology, Cat#9562), Hemagglutinin (HA) (Covance, Princeton, NJ; Cat#MMS-101P, www.covance.com ), Ran (BD Biosciences, San Jose, CA; Cat#610340, www.bdbiosciences.com ), Cnot1 (Protein-tech, Chicago, IL; Cat#14276-1-AP, www.ptglab.com ), Cnot2 (Proteintech, Cat#10313-1-AP).

Techniques: Transfection, Western Blot, Cell Culture, Expressing, Microarray, Over Expression, Reporter Assay, Quantitative RT-PCR

Journal: Stem cells (Dayton, Ohio)

Article Title: Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

doi: 10.1002/stem.1070

Figure Lengend Snippet: Silencing Cnot1, Cnot2, or Cnot3 led to mouse embryonic stem cell (ESC) differentiation. (A): Silencing Cnot1, Cnot2, or Cnot3 resulted in ESC differentiation based on the Oct4GiP reporter assay. Oct4GiP ESCs were transfected with indicated siRNAs (two different siR NAs for each CCr4-Not complex gene) in M15 medium and cultured for 4 days. The percentage of differentiated cells (% differentiation) was determined by measuring the percentage of green fluorescent protein-negative cells by fluorescence-activated cell sorting (FACS) at the end of the culture. (B): Expression of siRNA-resistant Cnot2 or Cnot3 rescued the differentiation caused by Cnot2 or Cnot3 knockdown, respectively. Oct4GiP cells or Oct4GiP cells expressing siRNA-resistant Cnot2 (Cnot2-Rescue) or Cnot3 (Cnot3-Rescue) were transfected with Control, Cnot1-siRNA1, Cnot2-siRNA2, or Cnot3-siRNA2, and the percentage of differentiated cells was determined by the Oct4GiP reporter assays. Note that Cnot2-Rescue cells were not able to rescue the differentiation caused by Cnot1 or Cnot3 silencing, and Cnot3-Rescue cells were not able to rescue Cnot1 or Cnot2 silencing. ***, p < .001. (C): Silencing Cnot1, Cnot2, or Cnot3 resulted in morphological changes and loss of alkaline phosphatase (AP) staining in ESCs. Oct4GiP cells were transfected with the indicated siRNAs and cultured in the M15 medium. Cells were stained with the AP staining kit and imaged 4 days after transfection. (D): Cnot1, Cnot2, or Cnot3 silencing led to downregulation of ESC marker and upregulation of differentiation markers. Oct4GiP cells were transfected with the indicated siRNAs and cultured in the M15 medium. Cells were harvested for quantitative real-time PCR (qRT-PCR) analysis 4 days after transfection. ESC marker: Oct4; differentiation markers: Cdx2, Eomes, Gata3, Hand1, and Krt8. (E): Cnot1, Cnot2, or Cnot3 silencing reduced cell proliferation or viability in 2i medium. Oct4GiP cells were transfected with control-siRNA (Control), Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), or Cnot3-siRNA2 (Cnot3-KD) and cul tured in 2i medium. Cell numbers were counted by FACS 4 days after transfection and normalized to control-transfected cells. (F): Cnot1, Cnot2, or Cnot3 silencing led to differentiation in 2i medium. Oct4GiP cells were transfected with indicated siRNAs and cultured in 2i medium. Cells were harvested for qRT-PCR analysis 4 days after transfection. (G): Expression of C-terminally HA-tagged Cnot2 (Cnot2-HA) in E14Tg2a cells. Expression of the exogenous Cnot2-HA was detected in Western blot with the HA-antibody, and Ran was used as a loading control. Expression of total (endogenous and exogenous) Cnot2 was determined by qPCR in wild-type E14Tg2a (E14) and Cnot2-HA expressing cells. The expression of the Cnot2-HA was estimated to be ∼2-fold of the endogenous Cnot2 on the mRNA level. (H): Identification of Cnot1 and Cnot3 in Cnot2-HA immunoprecipitation. HA-pull-down was carried out in E14Tg2a cells expressing Cnot2-HA. The presence of Cnot1, Cnot2-HA, and Cnot3 in the total lysate and pull-down sample (HA-beads) were detected by Western blot. Note that Oct4 was not detected in the pull down sample. As a negative control, protein-A beads were used in an independent pull-down. Abbreviations: HA, hemagglutinin; IP, immunoprecipitation; KD, knockdown.

Article Snippet: Primary antibodies used in this study were as follows Oct4 (Abcam, Cambridge, MA; Cat#AB19857, www.abcam.com ), Nanog (Millipore, Billerica, MA; Cat#AB9220, www.millipore.com ), Smad1-5-8-phospho-specific (Cell Signaling Technology, Beverly, MA; Cat#9512, www.cellsignal.com ), Smad1 (Cell Signaling Technology, Cat#9511), Stat3-S727-phosphospecific (Cell Signaling Technology, Cat#9134), Stat3Y705-phospho-specific (Cell Signaling Technology, Cat#9145), Stat3 (Cell Signaling Technology, Cat#9132), b-Catenin-Ser45-phospho-specific (Cell Signaling Technology, Cat#9564), b-Catenin (Cell Signaling Technology, Cat#9562), Hemagglutinin (HA) (Covance, Princeton, NJ; Cat#MMS-101P, www.covance.com ), Ran (BD Biosciences, San Jose, CA; Cat#610340, www.bdbiosciences.com ), Cnot1 (Protein-tech, Chicago, IL; Cat#14276-1-AP, www.ptglab.com ), Cnot2 (Proteintech, Cat#10313-1-AP).

Techniques: Reporter Assay, Transfection, Cell Culture, Fluorescence, FACS, Expressing, Staining, Marker, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Negative Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

doi: 10.1002/stem.1070

Figure Lengend Snippet: Cnot1, Cnot2, and Cnot3 are expressed in mouse ESCs and the inner cell mass (ICM) of mouse blastocysts. (A): Cnot gene expression in mouse ES, MEF, and TS cells. The expression of the Cnot genes was determined by quantitative real-time PCR (qRT-PCR), and the ESC-specific marker Oct4 was used as a reference. (B): Cnot gene expression during ESC differentiation induced by embryoid body formation, retinoid acid treatment, and LIF withdrawal. The expression of the Cnot genes was determined by qRT-PCR at the indicated time points. Oct4 was used as a reference. (C): Detection of Cnot1, Cnot2, and Cnot3 transcripts in mouse E3.5 embryo via whole-mount in situ hybridization. Sense probes were used as negative controls, and the antisense probes showed the predominant expression of Cnot1, Cnot2, and Cnot3 in the ICM. Abbreviations: ES, embryonic stem; MEF, mouse embryonic fibroblast; TS, trophoblast stem.

Article Snippet: Primary antibodies used in this study were as follows Oct4 (Abcam, Cambridge, MA; Cat#AB19857, www.abcam.com ), Nanog (Millipore, Billerica, MA; Cat#AB9220, www.millipore.com ), Smad1-5-8-phospho-specific (Cell Signaling Technology, Beverly, MA; Cat#9512, www.cellsignal.com ), Smad1 (Cell Signaling Technology, Cat#9511), Stat3-S727-phosphospecific (Cell Signaling Technology, Cat#9134), Stat3Y705-phospho-specific (Cell Signaling Technology, Cat#9145), Stat3 (Cell Signaling Technology, Cat#9132), b-Catenin-Ser45-phospho-specific (Cell Signaling Technology, Cat#9564), b-Catenin (Cell Signaling Technology, Cat#9562), Hemagglutinin (HA) (Covance, Princeton, NJ; Cat#MMS-101P, www.covance.com ), Ran (BD Biosciences, San Jose, CA; Cat#610340, www.bdbiosciences.com ), Cnot1 (Protein-tech, Chicago, IL; Cat#14276-1-AP, www.ptglab.com ), Cnot2 (Proteintech, Cat#10313-1-AP).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Marker, In Situ Hybridization

Journal: Stem cells (Dayton, Ohio)

Article Title: Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

doi: 10.1002/stem.1070

Figure Lengend Snippet: Cnot1, Cnot2, and Cnot3 are required for human embryonic stem cell (ESC) self-renewal. (A): Cnot1, Cnot2, and Cnot3 were down-regulated during human ESC differentiation. H1 human ESCs were differentiated for 7 days using 100 ng/ml human recombinant BMP4. The expression levels of Cnot1, Cnot2, and Cnot3 as well as Oct4 and differentiation markers Cdx2 and Hand1 were determined by quantitative realtime PCR (qRT-PCR). (B): Silencing of Cnot1, Cnot2, or Cnot3 led to morphological changes of human ESCs. H1 cells were imaged 6 days after transfection. Phase-contrast images highlight the undifferentiated morphology of human ESCs in the lipids-only transfected cells (mock) versus the differentiated phenotype in the Cnot1, Cnot2, or Cnot3 siRNA transfected cells. (C): Silencing of the Cnot genes led to upregulation of the Cdx2 and Gata3 proteins. H1 cells were transfected with lipids-only (mock), Oct4, Cnot2, or Cnot3 siRNAs. Cells were fixed and stained for Cdx2 or Gata3 expression by immunofluorescence staining 6 days after transfection. (D): Silencing of the Cnot genes led to downregulation of the ESC marker and upregulation of the extraembryonic markers. H1 cells were harvested 6 days after transfection and marker expression was determined by qRT-PCR. Abbreviations: BMP, bone morphogenetic protein; DAPI, 4′-6-diamidino-2-phenylindole.

Article Snippet: Primary antibodies used in this study were as follows Oct4 (Abcam, Cambridge, MA; Cat#AB19857, www.abcam.com ), Nanog (Millipore, Billerica, MA; Cat#AB9220, www.millipore.com ), Smad1-5-8-phospho-specific (Cell Signaling Technology, Beverly, MA; Cat#9512, www.cellsignal.com ), Smad1 (Cell Signaling Technology, Cat#9511), Stat3-S727-phosphospecific (Cell Signaling Technology, Cat#9134), Stat3Y705-phospho-specific (Cell Signaling Technology, Cat#9145), Stat3 (Cell Signaling Technology, Cat#9132), b-Catenin-Ser45-phospho-specific (Cell Signaling Technology, Cat#9564), b-Catenin (Cell Signaling Technology, Cat#9562), Hemagglutinin (HA) (Covance, Princeton, NJ; Cat#MMS-101P, www.covance.com ), Ran (BD Biosciences, San Jose, CA; Cat#610340, www.bdbiosciences.com ), Cnot1 (Protein-tech, Chicago, IL; Cat#14276-1-AP, www.ptglab.com ), Cnot2 (Proteintech, Cat#10313-1-AP).

Techniques: Recombinant, Expressing, Quantitative RT-PCR, Transfection, Staining, Immunofluorescence, Marker