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: 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: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 1 Regulation of PKN kinase activity. IP-kinase assays with WT and TM mutants of PKN1 (S916A) and PKN2 (T958A). Torin inhibited the PKN kinase activity to about the same extent as mutating the TM in both PKN isoforms. B, The PKN1 TM mutant S916A has reduced kinase activity toward multiple substrates. C, Deletion of the PKN N-terminus results in constitutive histone H3 phosphorylation in vitro and in cells. D and E, The PKN1 TM mutant S916A dramatically reduces autophosphorylation as well as Histone H3 and MARCKS phosphorylation

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Activity Assay, Mutagenesis, Phospho-proteomics, In Vitro

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 3 Torin and rapamycin sensitivity of PKN, AKT, and PKCα. A, Cells stably transduced with WT PKN1 were treated with a range of torin and rapamycin concentrations for 24 h, and analyzed by using pan- and phosphosite-specific antibodies. B, Cells were treated with torin and rapamycin during a time course up to 24 h and subsequently analyzed by using pan- and phosphosite- specific antibodies

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Stable Transfection, Transduction, Phospho-proteomics

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 2 PKN contains a TM phosphorylated by a torin- sensitive kinase. A, Alignment of TM sequences with the predicted phosphorylated residues indicated (bold). B, Transfection of PKN1 bearing mutations in the TM (S916A), activation loop (T774E) and ATP binding pocket (K644E) probed with antibodies specific for phos-S916 and phos-T774. Including nonphospho-TM peptide during the antibody incubation reduces the detection of non- phosphorylated PKN. C, IP-blot of WT PKN1 expressed in cells treated with torin and rapamycin

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Transfection, Activation Assay, Binding Assay, Incubation

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 4 Cell motility functions of PKN. A, Localization of Flag-tagged PKN1 (green) at the cleavage furrow during mitosis, imaged by confocal microscopy. B, Examples of binucleate cells generated in response to depletion of PKN1, PKN2, and Ect2 (positive control), indicative of cytokinesis failure. C, Quantification of cytokinesis failure data as a consequence of PKN1 and PKN2 depletion. D, Expression levels (immunoblotting) of PKN1 and PKN2 after siRNA depletion. E, Stable C4-2b cell lines showing that (E) ectopic expression and (F) knockdown increase and decrease, respectively, cell migration in a Boyden chamber assay (****P < 0.0001). G, Transient depletion of PKN1, PKN2, and the TORC2 subunit Rictor reduces cell invasion of PC-3 cells to a similar extent as torin treatment

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Confocal Microscopy, Generated, Positive Control, Expressing, Western Blot, Knockdown, Migration, Boyden Chamber Assay

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 5 Analysis of PKN isoform expression in human prostate cancer. A, Representative IHC showing PKN1 protein levels in normal, primary tumor, and lymph node metastasis. B, PKN1 and PKN2 expression (using microarray data from reference 47) in normal prostate, primary tumor, and metastases. C, RNA expression (using RNAseq data from TCGA) of PKN1-3 isoforms, PTEN, PKCα, AKT, and select mTOR components. **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Expressing, Microarray, RNA Expression

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 6 Pkn2 is required for embryonic development. A, Embryos from Pkn1 and Pkn2 lacZ reporter mice were stained for β- galactosidase activity, and are shown as whole mount images. Upper row: E10.5, E11.5, E11.5. Scale bars: 1.0 mm. Bottom row: E6.5, E8.5 (side and dorsal view), E9.5, E9.5. Scale bars: 0.2 mm, 0.5 mm, 1.0 mm. B, Whole mount images of Pkn2 heterozygotes and homozygous null embryos at E7.0, E7.75, and E9.5. Scale bars 0.2 mm (upper four panels) 1.0 mm. C, Whole mount images of wild-type and Pkn2 null embryos analyzed by whole mount in situ hybridization for Otx2 (E7.5) and Bra (E7.25) are shown. Scale bars: 0.2 mm

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Staining, Activity Assay, In Situ Hybridization

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 7 Analysis of PKN1 overexpression in prostate. A, Immunoblots showing transgenic expression of full-length (Tg-PKN1) and constitutively active (Tg-PKN1ΔN) proteins in anterior, dorsal, lateral, and ventral lobes (AP, DP, LP, VP). B-E, H&E stained images of sections through the ventral prostates from mice of the indicated genotypes are shown. The ages of the mice are as follows: WT, 53 weeks; Tg-PKN1, 58 weeks; Tg-PKN1ΔN, 58 weeks; Tg-AKT1, 52 weeks; Tg-AKT1;Tg-PKN1, 41 weeks; Tg-AKT1;Tg-PKN1ΔN, 52 weeks; TRAMP and TRAMP;Tg-PKN1, 16 weeks (showing HGPIN); TRAMP and TRAMP;Tg-PKN1, 17 weeks (showing small cell carcinoma). All images were captured at 200× magnification. Lower magnification views of the same samples are also provided (Supplemental Figure S3)

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Over Expression, Western Blot, Transgenic Assay, Expressing, Staining

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 8 Analysis of PKNs in Pten null prostate tumors. H&E stained images of sections through the prostates from mice of the indicated genotypes are shown. All images were captured at 200× magnification and are of the ventral prostate, except for the right-most image in panel D, which shows squamous differentiation from the anterior prostate. The ages of the mice (panels A–C) are as follows: Ptenr/r, 12 and 45 weeks; Ptenr/r;Tg-PKN1, 12 and 43 weeks; Ptenr/r;Pkn1r/r;Pkn2r/r, 26 and 45 weeks. D, The images of invasive cancer (left and middle) are from 53-week ventral prostates, the squamous differentiation shown to the right is from the anterior prostate of a 53-week animal. Lower magnification views of the same samples are also provided (Supplemental Figure S4)

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Staining

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: Cellular & Molecular Biology Letters

Article Title: Targeting LINC02320 prevents colorectal cancer growth via GRB7-dependent inhibition of MAPK signaling pathway

doi: 10.1186/s11658-025-00770-2

Figure Lengend Snippet: LINC02320 displays increased levels in CRC and is associated with poor prognosis. A The adjacent normal tissue, primary CRC tissue and metastatic tissue in the liver from the same patient were collected and subjected to total RNA isolation and RNA-sequencing (mRNA plus lncRNA). The genes (FC ≥ 2 and p < 0.05) that were upregulated both in primary tumor tissue versus paired adjacent normal tissue and in metastatic tumor tissue versus primary tumor tissue were presented as a heatmap. B The levels of the 5 most upregulated lncRNAs (LINC01811, LINC02418, LINC02577, MGC32805 and LINC02320) were analyzed using the UALCAN database ( https://ualcan.path.uab.edu/analysis.html ). C The expression patterns of lncRNAs as indicated in (B) were determined in CRC and adjacent normal tissues according to the online sequencing data ( GSE104836 ). D Twelve paired CRC and adjacent normal tissues were collected, followed by detection of LINC02320 level using RT-qPCR. E and F The adjacent normal tissue, paired primary CRC tissue and metastatic tissue in the liver were obtained from three independent patients and subjected to RT-qPCR analysis and in situ hybridization (ISH) staining for LINC02320 expression evaluation. Scale bar, 50 μm. G LINC02320 levels were quantified in tissue microarray (TMA) by ISH. Scale bar, 200 μm. H LINC02320 expression was determined in CRC patients with early stages (I/II) and advanced stages (III/IV) according to the TMA result. Scale bar, 200 μm. I The relationship between LINC02320 expression and prognosis was investigated. J RNA fluorescent in situ hybridization (FISH) was conducted to ascertain the expression of LINC02320 in HCT116 and SW480 cells. Scale bar, 10 μm. ns, not significant. * p < 0.05, ** p < 0.01 and *** p < 0.001. Data are presented as means ± SD. Data were analyzed by an unpaired Student’s t -test and were representative of at least three independent experiments

Article Snippet: Human colorectal cancer cell lines HCT116, HT29, SW480, LoVo and RKO were obtained from the American Type Culture Collection.

Techniques: Isolation, RNA Sequencing, Expressing, Sequencing, Quantitative RT-PCR, In Situ Hybridization, Staining, Microarray

Journal: Cellular & Molecular Biology Letters

Article Title: Targeting LINC02320 prevents colorectal cancer growth via GRB7-dependent inhibition of MAPK signaling pathway

doi: 10.1186/s11658-025-00770-2

Figure Lengend Snippet: Depletion of LINC02320 impedes tumor growth and metastasis both in vitro and in vivo. A The efficacy of LINC02320 knockdown was evaluated through qRT-PCR. NC, negative control. B and C CCK-8 and colony formation assays were conducted in HCT116 and SW480 cells following LINC02320 knockdown to assess cellular proliferation potential. NC, negative control. D and E Wound healing and transwell assays were employed to evaluate the migratory capacity of HCT116 and SW480 cells following LINC02320 depletion. NC, negative control. Scale bar, 100 μm. F Patient-derived organoids (PDOs) assay were performed after transfected with indicated shRNA and organoid viability was assessed. Scale bar, 100 μm. G and H Xenograft experiments were performed using shLINC02320 or shNC HCT116 stable cell lines. Tumor volumes were monitored every 3 days while tumor weights were measured at the conclusion of the experimental period. I Total RNA was extracted from tumor tissues of the xenograft assay and LINC02320 expression was analyzed by RT-qPCR. J and K Immunohistochemical (IHC) analysis was employed to test Ki67 and cleaved Caspase-3 protein levels in tumor tissues of the xenograft assay. Scale bar, 100 μm. L HCT116 cells (5 × 10 6 ) stably expressing shLINC02320 or shNC were injected into the 5-week-old nude mice in tail vein. 6 weeks later, the presence of metastatic nodules in the lung were assessed, with representative images of the lung presented. M and N The lungs with metastatic nodules were subjected to hematoxylin–eosin (HE) staining and the number of nodules was quantified. Scale bar, 1000 μm. shRNA indicates shLINC02320. * p < 0.05, ** p < 0.01 and *** p < 0.001. Data are presented as means ± SD. Data were analyzed by an unpaired Student’s t -test and are representative of at least three independent experiments for in vitro assays

Article Snippet: Human colorectal cancer cell lines HCT116, HT29, SW480, LoVo and RKO were obtained from the American Type Culture Collection.

Techniques: In Vitro, In Vivo, Knockdown, Quantitative RT-PCR, Negative Control, CCK-8 Assay, Derivative Assay, Transfection, shRNA, Stable Transfection, Xenograft Assay, Expressing, Immunohistochemical staining, Injection, Staining

Journal: Cellular & Molecular Biology Letters

Article Title: Targeting LINC02320 prevents colorectal cancer growth via GRB7-dependent inhibition of MAPK signaling pathway

doi: 10.1186/s11658-025-00770-2

Figure Lengend Snippet: LINC02320 activates the MAPK pathway in a GRB7-dependent manner. A Volcano plot based on the RNA sequencing (RNA-seq) of total RNAs from the shLINC02320 or shNC stable HCT116 cell line. ShNC represents the negative control shRNA. shLncRNA denotes the LINC02320 shRNA. B KEGG pathway analysis based on the differentially expressed genes in shLncRNA HCT116 cells versus shNC controls. C – E Gene Ontology (GO) analysis of molecular function, cell component and biological process according to the differentially expressed genes in shLncRNA HCT116 cells versus shNC controls. F Gene Set Enrichment Analysis (GSEA) plots indicating a positive correlation between LINC02320 level and epithelial-mesenchymal transition (EMT) signatures. G A combined analysis of the RNA-seq results and an online public data set ( GSE44076 ). The genes that were identified as intersecting are illustrated in a volcano diagram on the right. H Five potential candidates were selected and their expression was quantified by RT-qPCR in shLINC02320 or shNC HCT116 and SW480 cell lines. I MAPK pathway activation was measured by Western blot after LINC02320 overexpression. J Western blotting assay demonstrating that LINC02320 depletion inactivates the MAPK signaling pathway while GRB7 ectopic expression restored its activation. Data are presented as means ± SD and are representative of at least three independent experiments

Article Snippet: Human colorectal cancer cell lines HCT116, HT29, SW480, LoVo and RKO were obtained from the American Type Culture Collection.

Techniques: RNA Sequencing, Negative Control, shRNA, Expressing, Quantitative RT-PCR, Activation Assay, Western Blot, Over Expression

Journal: Cellular & Molecular Biology Letters

Article Title: Targeting LINC02320 prevents colorectal cancer growth via GRB7-dependent inhibition of MAPK signaling pathway

doi: 10.1186/s11658-025-00770-2

Figure Lengend Snippet: LINC02320 facilitates ILF2 recruitment to initiate GRB7 transcription. A Luciferase reporter assay showing that LINC02320 overexpression stimulates the transcription of GRB7 . B HCT116 cells were lysed, and the supernatant was precleared using streptavidin-coupled beads and then incubated with biotin-LINC02320 or biotin-scramble sequence, followed by enrichment with streptavidin-coupled beads. Subsequently, supernatant was subjected to SDS-PAGE, followed by silver staining. The differential band in the biotin-LINC02320 lane was identified by mass spectrometry (MS). C RNA pulldown using HCT116 or SW480 cell lysates and biotin-LINC02320 or biotin-scramble demonstrated the interaction between ILF2 and LINC02320. D RNA immunoprecipitation (RIP) using anti-ILF2 or IgG confirmed the binding of LINC02320 to ILF2. E RNA-FISH were conducted using Cy3-coupled LINC02320 probes and an ILF2 antibody. Scale bar, 10 μm. F LINC02320 truncations were constructed and domain mapping was performed to determine its interactive region with ILF2. G Luciferase reporter assay was conducted to examine the luciferase activity of the GRB7 promoter following the ectopic expression of ILF2. H CUT&Tag was performed to test the ILF2 enrichment at GRB7 locus after LINC02320 overexpression in HCT116 cells. I Luciferase reporter assay was performed to analyze the luciferase activity of the GRB7 promoter after overexpression of LINC02320 and ILF2. J The relative RNA levels of ILF2 and GRB7 were quantified through RT-qPCR in stable ILF2-silencing HCT116 and SW480 cell lines. K The relative RNA levels of ILF2 and GRB7 were evaluated via RT-qPCR in stable ILF2-overexpressing HCT116 and HT29 cell lines. L The activation of the MAPK pathway and GRB7 protein level were detected by western blotting in ILF2-overexpressing cell lines. M Correlation of GRB7 expression with the ILF2 level in CRC tissues from GSE44076 dataset by the Pearson correlation coefficient. *** p < 0.001. Data are presented as means ± SD. Data were analyzed by an unpaired Student’s t -test and are representative of at least three independent experiments

Article Snippet: Human colorectal cancer cell lines HCT116, HT29, SW480, LoVo and RKO were obtained from the American Type Culture Collection.

Techniques: Luciferase, Reporter Assay, Over Expression, Incubation, Sequencing, SDS Page, Silver Staining, Mass Spectrometry, RNA Immunoprecipitation, Binding Assay, Construct, Activity Assay, Expressing, Quantitative RT-PCR, Activation Assay, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: Targeting LINC02320 prevents colorectal cancer growth via GRB7-dependent inhibition of MAPK signaling pathway

doi: 10.1186/s11658-025-00770-2

Figure Lengend Snippet: LINC02320-GRB7-MAPK-FOS constitutes a positive feedback loop. A The relative RNA levels of GRB7 and LINC02320 were evaluated by RT-qPCR in HCT116 and SW480 cells stably expressing shGRB7 or shNC. shNC, negative control shRNA. B Relative expression of LINC02320 in HCT116 and HT29 cells with GRB7 overexpression and the addition of the MAPK inhibitor AZD6244 was evaluated. Vec, empty vector. C The luciferase activity of the LINC02320 promoter was examined. D The potential motifs for transcription factor (TF) binding in the LINC02320 promoter was analyzed through the JASPAR online tool. E The activation of FOS and ELK1 was measured following LIN02320 overexpression through the detection of phosphorylation in HCT116 and HT29 cells. F and G The activation of MAPK pathway was detected by Western blot after GRB7 knockdown or overexpression. H The luciferase activity of LINC02320 was evaluated after FOS overexpression. I CUT&Tag was performed to test the H3K27ac enrichment at LINC02320 locus after FOS overexpression in HCT116 cells. J FOS overexpression was validated by RT-qPCR. K Relative expression of LINC02320 was analyzed after FOS ectopic expression. *** p < 0.001. Data are presented as means ± SD. Data were analyzed by an unpaired Student’s t -test and are representative of at least three independent experiments

Article Snippet: Human colorectal cancer cell lines HCT116, HT29, SW480, LoVo and RKO were obtained from the American Type Culture Collection.

Techniques: Quantitative RT-PCR, Stable Transfection, Expressing, Negative Control, shRNA, Over Expression, Plasmid Preparation, Luciferase, Activity Assay, Binding Assay, Activation Assay, Phospho-proteomics, Western Blot, Knockdown

Journal: Cellular & Molecular Biology Letters

Article Title: Targeting LINC02320 prevents colorectal cancer growth via GRB7-dependent inhibition of MAPK signaling pathway

doi: 10.1186/s11658-025-00770-2

Figure Lengend Snippet: LINC02320 promotes CRC progression depending on the GRB7-MAPK signaling. A Disease-free survival rate of CRC patients was analyzed according to GRB7 expression using GEPIA database. B and C CCK-8 and colony formation assays were performed to test cell proliferation using the indicated cell lines. shNC, negative control shRNA. D and E Wound healing and transwell assays were used to measure cell migration. Scale bar, 100 μm. F HCT116 and HT29 cells were infected with LINC02320 overexpression virus and used for colony formation assays in the presence of AZD6244. Vec, empty vector. G Transwell assays was performed to determine the migratory ability of HCT116 and SW480 cells. Scale bar, 100 μm. H and I The xenograft experiment was conducted using 2 × 10 6 LINC02320-overexpressing HCT116 cells per mouse and nude mice were subjected to AZD6244 gavage. Then tumor volume was monitored and tumor weight was measured. J Tumor tissues from the xenograft experiment were fixed and utilized for analysis of Ki67 protein level through IHC staining. Scale bar, 50 μm. K and L Tumor volumes were measured every 2 days and tumor weights were determined at the end timepoint of the ASO targeting experiment. ns, not significant. * p < 0.05 and *** p < 0.001. Data are presented as means ± SD. Data were analyzed by an unpaired Student’s t -test and are representative of at least three independent experiments

Article Snippet: Human colorectal cancer cell lines HCT116, HT29, SW480, LoVo and RKO were obtained from the American Type Culture Collection.

Techniques: Expressing, CCK-8 Assay, Negative Control, shRNA, Migration, Infection, Over Expression, Virus, Plasmid Preparation, Immunohistochemistry

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

Journal: BMC Medical Genetics

Article Title: Cell type-specific over-expression of chromosome 21 genes in fibroblasts and fetal hearts with trisomy 21

doi: 10.1186/1471-2350-7-24

Figure Lengend Snippet: Differential gene expression in control vs. +21 fibroblasts . Results from supervised hierarchical clustering of the U95A microarray data are shown; with statistical criteria for selecting the genes indicated at the top (ANOVA, see Methods). Genes (probe sets) are on the x-axis and samples are on the y-axis, with expression indicated by the color scale from 0 (blue) to 5 (red), relative to the experiment mean. A, Data from early passage cells. As shown in the panel on the right, in which genes not on chromosome 21 have been blacked out, the set of over-expressed genes is enriched in genes on chromosome 21 and no genes on chromosome 21 are in the under-expressed set. B, Data from late passage cells. The MX1 gene is markedly over-expressed as the +21 fibroblasts become senescent. See Tables 2 and 3 for the complete lists of differentially expressed genes.

Article Snippet: After transferring to the nitrocellulose membrane and blocking by 5% milk, the membrane was hybridized with mouse antibodies against p78 MX1 or α-actin (Santa Cruz Biotechnology, Santa Cruz, CA) in a solution containing 0.1% BSA, 0.1% Tween-20, and 3% dry milk overnight at 4°C.

Techniques: Gene Expression, Control, Microarray, Expressing

Journal: BMC Medical Genetics

Article Title: Cell type-specific over-expression of chromosome 21 genes in fibroblasts and fetal hearts with trisomy 21

doi: 10.1186/1471-2350-7-24

Figure Lengend Snippet: Genes over-expressed in late passage +21 fibroblasts passing ANOVA at p < .01. Genes on chromosome 21 are in bold.

Article Snippet: After transferring to the nitrocellulose membrane and blocking by 5% milk, the membrane was hybridized with mouse antibodies against p78 MX1 or α-actin (Santa Cruz Biotechnology, Santa Cruz, CA) in a solution containing 0.1% BSA, 0.1% Tween-20, and 3% dry milk overnight at 4°C.

Techniques:

Journal: BMC Medical Genetics

Article Title: Cell type-specific over-expression of chromosome 21 genes in fibroblasts and fetal hearts with trisomy 21

doi: 10.1186/1471-2350-7-24

Figure Lengend Snippet: Over-expression of MX1 mRNA in fibroblasts with +21. A, Scatter plot of the GeneChip data for MX1 , showing the range of expression of this gene in +21 and control fibroblasts at early and late passage. The data in this figure are from the control and +21 fibroblast lines at the passage numbers and percentage of cells positive for acid beta-galactosidase listed in Table 1. B, northern blots showing the over-expression of MX1 mRNA, particularly with increasing passage, in a +21 fibroblast line (DS1) compared to a control fibroblast line (C1). Passage number is indicated above each lane. Re-hybridization with a GAPDH cDNA probe is shown as a loading control. The blots were stripped and re-hybridized with an IFI27 cDNA probe, indicating that this interferon-inducible gene also becomes over-expressed as the cells senesce; but with somewhat less differential expression than MX1 . Similar northern blotting results were obtained with two other control and two other +21 fibroblast lines (data not shown). C , Quantitation of the northern blot signals by Phosphorimaging shows increasing MX1 mRNA (normalized to GAPDH ) as a function of passage number in +21 fibroblasts, with a more modest increase in control fibroblasts.

Article Snippet: After transferring to the nitrocellulose membrane and blocking by 5% milk, the membrane was hybridized with mouse antibodies against p78 MX1 or α-actin (Santa Cruz Biotechnology, Santa Cruz, CA) in a solution containing 0.1% BSA, 0.1% Tween-20, and 3% dry milk overnight at 4°C.

Techniques: Over Expression, Expressing, Control, Northern Blot, Hybridization, Quantitative Proteomics, Quantitation Assay

Journal: BMC Medical Genetics

Article Title: Cell type-specific over-expression of chromosome 21 genes in fibroblasts and fetal hearts with trisomy 21

doi: 10.1186/1471-2350-7-24

Figure Lengend Snippet: Validation of p78 MX1 over-expression in +21 fibroblasts by western blotting, correlation of p78 MX1 protein with MX1 mRNA, and induction of MX1 mRNA in normal fibroblasts by interferon-beta present in +21 fibroblast-conditioned medium. A, p78 MX1 is over-expressed in +21 fibroblasts, directly correlating with MX1 RNA. The top two panels show results from a western blot of proteins from whole-cell lysates of +21 (n = 4) and control (n = 3) fibroblasts at late passage. The blot was re-probed with anti-beta-actin is as a loading control. The bottom panel shows the relative MX1 RNA levels of all the samples from the GeneChip data, normalized to the value for the late-passage C1 fibroblasts. P, passage number. The excess of MX1 mRNA in these cells leads to the expected excess production of p78 MX1 protein. B, conditioned medium from fibroblasts with trisomy 21 was added to the medium of control fibroblasts at the indicated proportions, either with our without the addition of anti-interferon beta antibodies. RNA was extracted after 24 hours and analyzed by northern blotting. Addition of anti-interferon almost completely abrogates the induction of MX1 mRNA by the +21 conditioned medium.

Article Snippet: After transferring to the nitrocellulose membrane and blocking by 5% milk, the membrane was hybridized with mouse antibodies against p78 MX1 or α-actin (Santa Cruz Biotechnology, Santa Cruz, CA) in a solution containing 0.1% BSA, 0.1% Tween-20, and 3% dry milk overnight at 4°C.

Techniques: Biomarker Discovery, Over Expression, Western Blot, Control, Northern Blot

Journal: BMC Medical Genetics

Article Title: Cell type-specific over-expression of chromosome 21 genes in fibroblasts and fetal hearts with trisomy 21

doi: 10.1186/1471-2350-7-24

Figure Lengend Snippet: Activation of p78 MX1 in lymphocytes and epithelial cells in a case of alopecia areata. A, low power field of a scalp biopsy with alopecia areata, showing an inflamed hair follicle (arrow) (H&E). B, low power field of staining with anti-p78 MX1 showing positive cells in and around the inflamed hair follicle (solid arrow), but not in an uninvolved region of another hair follicle (dashed arrow). Capillary endothelial cells stain throughout the dermis (asterisks). C, high power field of staining with anti-p78 MX1 , showing that the p78-positive cells are a subset of lymphocytes (arrow) and outer root sheath epithelial cells (ORS, lines), as well as matrix cells (M) around the dermal papilla (DP). The inner root sheath (IRS) does not stain. A second case of sporadic alopecia areata revealed a similar pattern of staining (data not shown). CTS = connective tissue sheath.

Article Snippet: After transferring to the nitrocellulose membrane and blocking by 5% milk, the membrane was hybridized with mouse antibodies against p78 MX1 or α-actin (Santa Cruz Biotechnology, Santa Cruz, CA) in a solution containing 0.1% BSA, 0.1% Tween-20, and 3% dry milk overnight at 4°C.

Techniques: Activation Assay, Staining

Journal: Molecular Cancer

Article Title: Circular RNA circTADA2A promotes osteosarcoma progression and metastasis by sponging miR-203a-3p and regulating CREB3 expression

doi: 10.1186/s12943-019-1007-1

Figure Lengend Snippet: The validation and expression of circTADA2A in osteosarcoma tissues and cells. a CircRNA microarray based on osteosarcoma cell lines and hFOB1.19 in GSE96964. b The expression of circTADA2A was detected by qRT-PCR in 10 osteosarcoma and chondroma tissues ( n = 10) (* P < 0.01, Student’s t -test). c Representative FISH images demonstrating circTADA2A expression detected by a junction probe in chondroma and osteosarcoma tissues; scale bars, 200 μm and 50 μm (FISH, fluorescence in situ hybridization). d CircTADA2A expression was detected by qRT-PCR in various human osteosarcoma cell lines (HOS, 143B, U2OS, SJSA-1 and MG63) and normal cells (osteoblast hFOB1.19 and HEK-293); circTADA2A mRNA levels were higher in OS cells than in hFOB1.19 and HEK-293 cells. e Schematic illustration demonstrates the formation of circTADA2A via the circularization of exons 5 and 6 in TADA2A (black arrow). The presence of circTADA2A was validated by RT-PCR, followed by Sanger sequencing. The head-to-tail splicing site of circTADA2A is indicated by the red arrow. f RT-PCR validated the existence of circTADA2A in HOS and 143B cell lines. CircTADA2A was amplified by divergent primers in cDNA but not gDNA. GAPDH was used as a negative control. g & h The expression of circTADA2A and TADA2A mRNA in both HOS and 143B cell lines was detected by RT-PCR or qRT-PCR in the presence or absence of RNase R. i FISH showed that circTADA2A was predominantly localized in the cytoplasm. Nuclei were stained with DAPI, and circTADA2A probes were labeled with Alexa Fluor 555; scale bars, 50 μm. Data are from three independent experiments (mean ± SEM) ( d and h ) or are representative of three independent experiments with similar results ( c , f , g and i ) (* P < 0.01 vs control or as indicated by Student’s t- test)

Article Snippet: HEK-293 (ATCC: CRL-1573), human osteoblast hFOB1.19 (ATCC: CRL-11372) and human OS cells, including the HOS (ATCC: CRL-1543), 143B (ATCC: CRL-8303), MG-63 (ATCC: CRL-1427TM), U2OS (ATCC: HTB-96TM) and SJSA-1 (ATCC: CRL-2098) cell lines, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Biomarker Discovery, Expressing, Microarray, Quantitative RT-PCR, Fluorescence, In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction, Sequencing, Amplification, Negative Control, Staining, Labeling, Control

Journal: Molecular Cancer

Article Title: Circular RNA circTADA2A promotes osteosarcoma progression and metastasis by sponging miR-203a-3p and regulating CREB3 expression

doi: 10.1186/s12943-019-1007-1

Figure Lengend Snippet: MiR-203a-3p regulates the migration, invasion and proliferation of OS cells. a The expression of miR-203a-3p was detected by qRT-PCR assay in 10 osteosarcoma and chondroma tissues ( n = 10) (* P < 0.01, Student’s t -test). b FISH showed the miR-203a-3p expression level was lower in osteosarcoma tissue than in chondroma tissue; scale bars, 200 μm and 50 μm. c MiR-203a-3p expression was detected by qRT-PCR in various human osteosarcoma cell lines (HOS, 143B, U2OS, SJSA-1 and MG63) and normal cells (osteoblast hFOB1.19 and HEK-293). d The expression of miR-203a-3p in OS at different clinicopathological stages was analyzed by FISH on an OS tissue chip (56 cases). Intensity of miR-203a-3p expression was calculated from the OS tissue chip. e The effect of miR-203a-3p on cell migration and invasion was evaluated by Transwell migration and Matrigel invasion assay. Representative images were shown. Scale bars, 100 μm. f Representative images of the wound-healing assay exhibiting changes in the migration capacity of stable OS cells. g Proliferation ability of stably transfected OS cells was evaluated by colony formation assay. Representative images are shown. h pre-miR-203a-3p (or miR-203a-3p sponge)-mediated miR-203a-3p overexpression (or knockdown) influenced the OS cell viability, as determined by a CCK-8 assay. Data are from three independent experiments (mean ± SEM) ( c, e-h ) or are representative of three independent experiments with similar results ( b ) (* P < 0.01 vs control or as indicated by Student’s t-test)

Article Snippet: HEK-293 (ATCC: CRL-1573), human osteoblast hFOB1.19 (ATCC: CRL-11372) and human OS cells, including the HOS (ATCC: CRL-1543), 143B (ATCC: CRL-8303), MG-63 (ATCC: CRL-1427TM), U2OS (ATCC: HTB-96TM) and SJSA-1 (ATCC: CRL-2098) cell lines, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Migration, Expressing, Quantitative RT-PCR, Invasion Assay, Wound Healing Assay, Stable Transfection, Transfection, Colony Assay, Over Expression, Knockdown, CCK-8 Assay, 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: Silencing Cnot1, Cnot2, or Cnot3 induce differentiation primarily into the TE lineage. (A): Cnot1, Cnot2, or Cnot3 knockdown induced similar gene expression changes. Venn diagram of genes that showed 1.5-fold changes after Cnot1, Cnot2, or Cnot3 knockdown. (B): Heatmap of expression changes for genes that are commonly affected by Cnot1, Cnot2, and Cnot3 knockdown. (C): Cnot1, Cnot2, or Cnot3 knockdown primarily led to TE differentiation. Principal component analysis (PCA) of embryonic stem cell (ESC) differentiation into three different lineages: pluripotent cells (gray spheres), TE differentiation time course and TE cells (blue spheres), PE differentiation time course (green spheres), and neural ectoderm differentiation time course (orange spheres). The three lineage-specific differentiation time courses form a tripod like structure in the PCA space (represented by the arrows). Cnot1 (C1), Cnot2 (C2), or Cnot3 (C3) silencing (red spheres) clustered closely to TE cells (blue). (D): Cdx2 overexpression and Cnot1, Cnot2, or Cnot3 knockdown resulted in similar gene expression changes. Two-dimensional matrix and heat map depicting gene expression changes in Cdx2-overexpression (Cdx2-OE) and Cnot1 (Cnot1-KD), Cnot2 (Cnot2-KD), or Cnot3 (Cnot3-KD) knockdowns, compared with control ESCs. Axes indicate degree of fold change, from nil (middle of axis) to greater than 1.5-fold (outermost square). Numbers indicate the median fold change of genes in each column or row. The intensity of each square represents the number of genes that fall in that square. The Pearson's correlation coefficients for the plots are: 0.24 for Cnot1-KD versus Cdx2-OE, 0.22 for Cnot2-KD versus Cdx2-OE, and 0.34 for Cnot3-KD versus Cdx2-OE. Abbreviations: KD, knockdown; NE, neuroectoderm; PE, primitive endoderm; TE, trophectoderm; 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: Knockdown, Gene Expression, Expressing, Over Expression, 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: 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: Knockdown, Transfection, Control, Western Blot, Cell Culture, Gene Expression, Microarray, Over Expression, Reporter Assay, Expressing, 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, Knockdown, Control, 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: Gene Expression, 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

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: Proposed model of Cnot1, Cnot2, and Cnot3 in ESC maintenance. Cnot1, Cnot2, and Cnot3 maintain mouse and human ESC identity and self-renewal by inhibiting differentiation into the extraembryonic lineages. Abbreviations: BMP, bone morphogenetic protein; ESC, embryonic stem cell; FGF, fibroblast growth factor; LIF, leukemia inhibitory factor.

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:

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a. TMEM180 gene expression in five CRC cell lines and two colonocyte samples from healthy donors was evaluated using DNA microarray analysis. b. TMEM180 gene expression in five clinical samples was evaluated using quantitative RT-PCR. Relative quantification as tumour-to-normal tissue ratio. c and d. ISH ( in situ hybridization) of TMEM180 in the clinical samples. Arrows indicate cancer (d).

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Expressing, Microarray, Quantitative RT-PCR, In Situ Hybridization

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a. Diagram of TMEM180 as a cation symporter is shown. b. Flow cytometric analysis of the SW480 wild-type (WT), gene knockdown clone No. 1 (KD1) or gene knockdown clone No. 2 (KD2) cells. The difference in the TMEM180 expression level in SW480-WT, -KD1 and -KD2 cells is shown. c. Cell growth of SW48-WT, -KD1 or -KD2 cells in (1) glutamine (Gln)–positive (+), arginine (Arg) (+) and Serum (+), (2) Gln (+), Arg (+) and Serum-negative (-), (3) Gln (+), Arg (-) and Serum (-) or (4) Gln (-), Arg (-) and Serum (-). Each growth activity was evaluated by WST-8 assay. d. Immunohistochemistry showing the expression of TMEM180 or EGFR in CRC and normal tissues (colon, brain, heart, lung, liver, kidney and skin). Scale bar = 100 μm.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Expressing, Activity Assay, Immunohistochemistry

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a. Purification of the TMEM180 protein from DLD-1 cells for the immunization. Gel filtration chromatography showed that a high concentration of TMEM180 protein existed in the void fraction with a molecular weight over 600 kDa. b. Immunogold electron microscopy. TMEM180 expression in tumour exosomes was determined. CD9 and CD63 were used as positive controls of standard exosome markers. Scale bar = 100 nm. c. Sandwich ELISA. Both TMEM180-positive and CD9 positive exosomes derived from DLD1 cells were determined. K562 cells were used as negative control. ***P < 0.001. Bar = SD.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Purification, Filtration, Chromatography, Concentration Assay, Molecular Weight, Electron Microscopy, Expressing, Sandwich ELISA, Derivative Assay, Negative Control

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a and b. Flow cytometric analysis of the colorectal cancer (CRC) cell lines DLD-1, HCT15, HCT116, HT29, WiDr and Colo320 (a). Flow cytometric analysis of haematopoietic cell lines Ramos, RL, Raji and K562 (b). DLD-1 was used as a positive control for TMEM180 expression.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Positive Control, Expressing

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a. Representative immunostaining patterns of TMEM180 and EGFR in CRC tissues were classified as negative (-), a weak positive (+) and strong positive (++). b. The number of cases with each expression level (-), (+) or (++) of TMEM180 or EGFR in CRC tissues (total number 37) was shown.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Immunostaining, Expressing

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a. Representative immunostaining patterns of TMEM180 and EGFR in CRC tissues were classified as negative (-), a weak positive (+) and strong positive (++). b. The number of cases with each expression level (-), (+) or (++) of TMEM180 or EGFR in CRC tissues (total number 37) was shown.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Immunostaining, Expressing

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a. In vitro cytotoxicity of the anti-TMEM180 mAb for CRC DLD-1 or SW480 cells in the WST-8 assay. The anti-TMEM180 mAb (=TMEM180), the anti-EGFR mAb cetuximab (=EGFR) or saline (=PBS) as a control was given at a concentration of 30 or 100 μg/ml. The percentage of surviving cells was calculated as the ratio of the number of cells in each group to that in the group given PBS treatment. b and c. ADCC (antibody-dependent cellular cytotoxicity, b) or CDC (complement-dependent cytotoxicity, c) activity of the anti-TMEM180 mAb for CRC DLD-1 cells, CRC SW480 cells and haematopoietic K562 cells. Red and blue lines indicate the ADCC or CDC activity of the anti-TMEM180 mAb (=TMEM180) and anti-EGFR mAb cetuximab (=EGFR), respectively. Bar = SD.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: In Vitro, Concentration Assay, Activity Assay

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a and b. Anti-tumour activity of the anti-TMEM180 mAb against DLD-1 (a) and SW480 (b) xenograft models. When the tumour volume reached approximately 200 mm , each treatment started (day 0). The anti-TMEM180 mAb (=TMEM180), the anti-EGFR mAb cetuximab (=EGFR) or saline (=PBS) as a control was administered at a dose of 12.5 or 25 mg/kg to each group of mice (n = 5) by intraperitoneal injection twice a week from Day 0 to 22. The arrows indicate the days of administration, and the curves show the effect of the treatment on tumour size. P < 0.001 (SW480 xenograft, PBS compared with TMEM180 12.5 mg/kg or 25 mg/kg; EGFR 12.5 mg/kg compared with TMEM180 25 mg/kg), P < 0.01 (DLD-1 xenograft, PBS compared with EGFR 25 mg/kg, TMEM180 12.5 mg/kg or 25 mg/kg; SW480 xenograft, EGFR 25 mg/kg compared with TMEM180 12.5 mg/kg). P < 0.05 (DLD-1 xenograft, PBS compared with EGFR 12.5 mg/kg). Bar = SD. c and d. Percent change in body weight in the same mice with the same treatments as in a and b. Bar = SD.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Activity Assay, Injection

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a. Schema of the promoter in the TMEM180 gene. Ten hypoxia response element consensus sequences (HREs) were found in the promoter sequence. HREs are indicated by arrows. b. Western blotting of HIF-1α. The difference in the HIF-1α protein expression level in the colorectal cancer (CRC) cell lines DLD-1 and SW480 under normoxia (21% O 2 ) and hypoxia (1% O 2 ) is shown. The expression of β-Actin was used as a control. c. Quantitative RT-PCR of TMEM180 . The difference in the TMEM180 mRNA expression level in DLD-1 and SW480 cells under normoxia (21% O 2 ) and hypoxia (1% O 2 ) is shown. **P < 0.01. Bar = SD. d and e. Immunostaining of TMEM180. The change in the TMEM180 expression pattern in DLD-1 and SW480 cells under normoxia (21% O 2 ) and hypoxia (1% O 2 ) is shown (D). Scale bar = 10 μm. The data were quantified and are shown in (E). ***P < 0.001. Bar = SD.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Sequencing, Western Blot, Expressing, Quantitative RT-PCR, Immunostaining

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a.Immunostaining of TMEM180-positive CRC cells with or without the tumour niche. αSMA (green), TMEM180 (red), Integrin α6 (white) and nuclei (blue) of a DLD-1 tumour and SW480 tumour are shown. Scale bar = 100 μm. Right, higher magnification of boxes area. Scale bar = 20 μm. b. In vitro colony-forming activity of SW480-WT, -KD1 and -KD2 cells. Representative images are shown (left). Scale bar = 50 μm. The number of colonies greater than 50 μm in diameter was counted (right). **P < 0.01. Bar = SD. c. CSC marker CD44 expression in SW480-WT and –KD1 and –kD2 cells. The representative expression level of CD44 using flow cytometry was shown. d. In vivo tumour initiation activity of SW480-WT, -KD1 and -KD2 cells was measured by evaluating the tumour incidence at the implanted sites on BALB/c nude mice 6 weeks after injection.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Immunostaining, In Vitro, Activity Assay, Marker, Expressing, Flow Cytometry, In Vivo, Injection

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a. TMEM180, EGFR, LGR5 and CD44 mRNA expression in ten normal tissues (brain, blood, connective, reproductive, muscular, alimentary, liver, lung, urinary and endo/exocrine tissue). Dataset was obtained from RefEx ( http://refex.dbcls.jp/ ). b. Comparison of TMEM180, EGFR, LGR5 and CD44 mRNA expression between colorectal cancer (N = 286) and normal tissue (N = 41). Dataset was obtained from UALCAN ( http://ulcan.path.uab.edu/index.html ).

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Expressing

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a. TMEM180 gene expression in five CRC cell lines and two colonocyte samples from healthy donors was evaluated using DNA microarray analysis. b. TMEM180 gene expression in five clinical samples was evaluated using quantitative RT-PCR. Relative quantification as tumour-to-normal tissue ratio. c and d. ISH ( in situ hybridization) of TMEM180 in the clinical samples. Arrows indicate cancer (d).

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Expressing, Microarray, Quantitative RT-PCR, In Situ Hybridization

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a. Diagram of TMEM180 as a cation symporter is shown. b. Flow cytometric analysis of the SW480 wild-type (WT), gene knockdown clone No. 1 (KD1) or gene knockdown clone No. 2 (KD2) cells. The difference in the TMEM180 expression level in SW480-WT, -KD1 and -KD2 cells is shown. c. Cell growth of SW48-WT, -KD1 or -KD2 cells in (1) glutamine (Gln)–positive (+), arginine (Arg) (+) and Serum (+), (2) Gln (+), Arg (+) and Serum-negative (-), (3) Gln (+), Arg (-) and Serum (-) or (4) Gln (-), Arg (-) and Serum (-). Each growth activity was evaluated by WST-8 assay. d. Immunohistochemistry showing the expression of TMEM180 or EGFR in CRC and normal tissues (colon, brain, heart, lung, liver, kidney and skin). Scale bar = 100 μm.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Expressing, Activity Assay, Immunohistochemistry

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a. Purification of the TMEM180 protein from DLD-1 cells for the immunization. Gel filtration chromatography showed that a high concentration of TMEM180 protein existed in the void fraction with a molecular weight over 600 kDa. b. Immunogold electron microscopy. TMEM180 expression in tumour exosomes was determined. CD9 and CD63 were used as positive controls of standard exosome markers. Scale bar = 100 nm. c. Sandwich ELISA. Both TMEM180-positive and CD9 positive exosomes derived from DLD1 cells were determined. K562 cells were used as negative control. ***P < 0.001. Bar = SD.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Purification, Filtration, Chromatography, Concentration Assay, Molecular Weight, Electron Microscopy, Expressing, Sandwich ELISA, Derivative Assay, Negative Control

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a and b. Flow cytometric analysis of the colorectal cancer (CRC) cell lines DLD-1, HCT15, HCT116, HT29, WiDr and Colo320 (a). Flow cytometric analysis of haematopoietic cell lines Ramos, RL, Raji and K562 (b). DLD-1 was used as a positive control for TMEM180 expression.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Positive Control, Expressing

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a. Representative immunostaining patterns of TMEM180 and EGFR in CRC tissues were classified as negative (-), a weak positive (+) and strong positive (++). b. The number of cases with each expression level (-), (+) or (++) of TMEM180 or EGFR in CRC tissues (total number 37) was shown.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Immunostaining, Expressing

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a. Representative immunostaining patterns of TMEM180 and EGFR in CRC tissues were classified as negative (-), a weak positive (+) and strong positive (++). b. The number of cases with each expression level (-), (+) or (++) of TMEM180 or EGFR in CRC tissues (total number 37) was shown.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Immunostaining, Expressing

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a. In vitro cytotoxicity of the anti-TMEM180 mAb for CRC DLD-1 or SW480 cells in the WST-8 assay. The anti-TMEM180 mAb (=TMEM180), the anti-EGFR mAb cetuximab (=EGFR) or saline (=PBS) as a control was given at a concentration of 30 or 100 μg/ml. The percentage of surviving cells was calculated as the ratio of the number of cells in each group to that in the group given PBS treatment. b and c. ADCC (antibody-dependent cellular cytotoxicity, b) or CDC (complement-dependent cytotoxicity, c) activity of the anti-TMEM180 mAb for CRC DLD-1 cells, CRC SW480 cells and haematopoietic K562 cells. Red and blue lines indicate the ADCC or CDC activity of the anti-TMEM180 mAb (=TMEM180) and anti-EGFR mAb cetuximab (=EGFR), respectively. Bar = SD.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: In Vitro, Concentration Assay, Activity Assay

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a and b. Anti-tumour activity of the anti-TMEM180 mAb against DLD-1 (a) and SW480 (b) xenograft models. When the tumour volume reached approximately 200 mm , each treatment started (day 0). The anti-TMEM180 mAb (=TMEM180), the anti-EGFR mAb cetuximab (=EGFR) or saline (=PBS) as a control was administered at a dose of 12.5 or 25 mg/kg to each group of mice (n = 5) by intraperitoneal injection twice a week from Day 0 to 22. The arrows indicate the days of administration, and the curves show the effect of the treatment on tumour size. P < 0.001 (SW480 xenograft, PBS compared with TMEM180 12.5 mg/kg or 25 mg/kg; EGFR 12.5 mg/kg compared with TMEM180 25 mg/kg), P < 0.01 (DLD-1 xenograft, PBS compared with EGFR 25 mg/kg, TMEM180 12.5 mg/kg or 25 mg/kg; SW480 xenograft, EGFR 25 mg/kg compared with TMEM180 12.5 mg/kg). P < 0.05 (DLD-1 xenograft, PBS compared with EGFR 12.5 mg/kg). Bar = SD. c and d. Percent change in body weight in the same mice with the same treatments as in a and b. Bar = SD.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Activity Assay, Injection

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a. Schema of the promoter in the TMEM180 gene. Ten hypoxia response element consensus sequences (HREs) were found in the promoter sequence. HREs are indicated by arrows. b. Western blotting of HIF-1α. The difference in the HIF-1α protein expression level in the colorectal cancer (CRC) cell lines DLD-1 and SW480 under normoxia (21% O 2 ) and hypoxia (1% O 2 ) is shown. The expression of β-Actin was used as a control. c. Quantitative RT-PCR of TMEM180 . The difference in the TMEM180 mRNA expression level in DLD-1 and SW480 cells under normoxia (21% O 2 ) and hypoxia (1% O 2 ) is shown. **P < 0.01. Bar = SD. d and e. Immunostaining of TMEM180. The change in the TMEM180 expression pattern in DLD-1 and SW480 cells under normoxia (21% O 2 ) and hypoxia (1% O 2 ) is shown (D). Scale bar = 10 μm. The data were quantified and are shown in (E). ***P < 0.001. Bar = SD.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Sequencing, Western Blot, Expressing, Quantitative RT-PCR, Immunostaining

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a.Immunostaining of TMEM180-positive CRC cells with or without the tumour niche. αSMA (green), TMEM180 (red), Integrin α6 (white) and nuclei (blue) of a DLD-1 tumour and SW480 tumour are shown. Scale bar = 100 μm. Right, higher magnification of boxes area. Scale bar = 20 μm. b. In vitro colony-forming activity of SW480-WT, -KD1 and -KD2 cells. Representative images are shown (left). Scale bar = 50 μm. The number of colonies greater than 50 μm in diameter was counted (right). **P < 0.01. Bar = SD. c. CSC marker CD44 expression in SW480-WT and –KD1 and –kD2 cells. The representative expression level of CD44 using flow cytometry was shown. d. In vivo tumour initiation activity of SW480-WT, -KD1 and -KD2 cells was measured by evaluating the tumour incidence at the implanted sites on BALB/c nude mice 6 weeks after injection.

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Immunostaining, In Vitro, Activity Assay, Marker, Expressing, Flow Cytometry, In Vivo, Injection

Journal: bioRxiv

Article Title: Significant antitumour effect of an antibody against TMEM180, a new cancer-specific molecule

doi: 10.1101/397844

Figure Lengend Snippet: a. TMEM180, EGFR, LGR5 and CD44 mRNA expression in ten normal tissues (brain, blood, connective, reproductive, muscular, alimentary, liver, lung, urinary and endo/exocrine tissue). Dataset was obtained from RefEx ( http://refex.dbcls.jp/ ). b. Comparison of TMEM180, EGFR, LGR5 and CD44 mRNA expression between colorectal cancer (N = 286) and normal tissue (N = 41). Dataset was obtained from UALCAN ( http://ulcan.path.uab.edu/index.html ).

Article Snippet: For the overexpression of TMEM180 gene, plasmid pCMV-TMEM180 (Origene) was transfected into DLD-1 cells (DLD-1-OE) using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacture’s protocol.

Techniques: Expressing