Journal: Aging and Disease

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

doi: 10.14336/AD.2022.0826

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

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

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

Journal: Aging and Disease

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

doi: 10.14336/AD.2022.0826

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

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

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

Journal: Aging and Disease

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

doi: 10.14336/AD.2022.0826

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

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

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

Journal: Aging and Disease

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

doi: 10.14336/AD.2022.0826

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

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

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

Journal: Aging and Disease

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

doi: 10.14336/AD.2022.0826

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

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

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

Journal: eLife

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

doi: 10.7554/eLife.58215

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

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

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

Journal: eLife

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

doi: 10.7554/eLife.58215

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

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

Techniques: Electroporation, Mutagenesis

Journal: eLife

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

doi: 10.7554/eLife.58215

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

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

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

Journal: eLife

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

doi: 10.7554/eLife.58215

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

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

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

Journal: eLife

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

doi: 10.7554/eLife.58215

Figure Lengend Snippet:

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

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

Journal: The Journal of Biological Chemistry

Article Title: Synthesis of Mitochondrial DNA Precursors during Myogenesis, an Analysis in Purified C2C12 Myotubes *

doi: 10.1074/jbc.M112.441147

Figure Lengend Snippet: Microarray analysis of genes regulating the dNTP pools in muscle cells. Samples were as follows: myoblasts ( Mb ), proliferating C2C12 myoblasts at 50% confluence; Myt , myotubes purified at 8 days of differentiation; EDL , extensor digitorum longus; and soleus ( SOL ), skeletal muscles dissected from adult CD1 mice. To measure expression levels, Cy3-labeled target from myoblasts, extensor digitorum longus and soleus preparations were hybridized in triplicate experiments on whole mouse genome oligonucleotide microarrays (4 × 44,000, Agilent Technologies). Normalized values were converted to log2 (scale on the left ), and heat maps were constructed with MultiExperiment Viewer to visualize gene expression levels. All genes involved in dNTP metabolism were annotated on the basis of their cytosolic or mitochondrial localization ( supplemental Table 1 ). To evaluate the significance of the expression changes, pairwise Significance Analysis of Microarrays two class analyses were carried out between myoblasts and each of the other samples. A , genes differentially expressed in at least one test using a false discovery rate of 1%. B , genes that showed no significant changes in muscle fibers compared with myoblasts. C , genes from A , sorted according to fold-change values to underline the predominant down-regulation observed in comparisons between cycling and differentiated muscle cells. A double asterisk marks genes with the most significant changes (false discovery rate, 0% in at least two tests). The inset shows the few genes with positive fold-changes.

Article Snippet: Incubation proceeded for 17 h at 65 °C at 10 rpm in a microarray hybridization oven (Agilent Technologies).

Techniques: Microarray, Purification, Expressing, Labeling, Construct

Journal: Nature protocols

Article Title: Array painting: a protocol for the rapid analysis of aberrant chromosomes using DNA microarrays

doi: 10.1038/nprot.2009.183

Figure Lengend Snippet: A schematic outlining the steps of an array paint experiment. (a) Derivative chromosomes are isolated from a cell line by chromosome sorting (Steps 1–22), the derivative chromosomes are then amplified using a GenomePlex Complete Whole Genome Amplification Kit, (Steps 23–38) and the derivative chromosome amplified DNA is fluorescently labeled in Cy3 or Cy5 (Steps 39–54). (b) The fluorescently labeled derivative DNA is hybridized onto a microarray using Agilent SureHyb chamber (Steps 55–80) (picture courtesy of Agilent). (c) The microarray is scanned (Steps 81–83). (d) The features on the microarray are analyzed, the log2 ratio of Cy5/Cy3 intensity is calculated for each oligonucleotide and plotted against the base-pair chromosome position (Steps 84–85). The top schematic represents the data for one parent chromosome (chr A), whereas the lower schematic represents the other parent chromosome (chr B). The transition from a high (red spots) to a low log2 ratio (green spots) indicates a chromosome breakpoint. If a feature sequence spans a chromosome breakpoint, the ratio will be close to 0 (orange spots).

Article Snippet: Agilent aCGH Wash Buffer 1 (Agilent, cat. no. 5188-5221) Agilent aCGH Wash Buffer 2 (Agilent, cat. no. 5188-5222) EQUIPMENT 0.22-μm syringe filter (Nalgene, cat. no. 190-2520) 20-μm mesh filter (Celltrics, Partec, cat. no. 04-004-2325) MoFlo High Performance Cell Sorter equipped with two water-cooled lasers (Coherent, Innova 300 series) 1.5-ml microfuge tubes Benchtop microfuge Heat block Thermal cycler Microcon YM-30, 30 kDa, sample reservoirs and tubes (Millipore, cat. no. 42422) NanoDrop 2000 Spectrophotometer (Thermo Scientific) Hybridization Gasket Slide Kit (5) for 1 microarray per slide format (Agilent, cat. no. G2534-60003) Hybridization Chamber Kit (Agilent, cat. no. G2534A) Blunt forceps (supplied with hybridization chamber) Human Genome CGH Microarray Kit 244A, consists of 5 microarrays (Agilent, cat. no. G4411B) DNA Microarray Hybridization Oven (Agilent, cat. no. G2545A) set to 65 °C, 20 rpm Hybridization Oven Rotator Rack (Agilent, cat. no. G2530-60029) Three glass troughs One slide rack Microarray Scanner (we used Agilent G2565CA but G2565BA can be used, Agilent) Slide holders supplied with scanner Feature Extraction 9.5 or 10.5 software and Agilent Scan Control software—both supplied with scanner DNA Analytics 4.0 (Agilent, cat. no. G4172AA) Freely available ‘R’ software Summit v3.1 (analysis software from Dako/Beckman Coulter) REAGENT SETUP Cell culture medium Supplement 500 ml of RMPI 1640 or DMEM with 15% (vol/vol) fetal calf serum and antibiotics.

Techniques: Isolation, Amplification, Whole Genome Amplification, Labeling, Microarray, Sequencing

Journal: Nature protocols

Article Title: Array painting: a protocol for the rapid analysis of aberrant chromosomes using DNA microarrays

doi: 10.1038/nprot.2009.183

Figure Lengend Snippet: Troubleshooting table.

Article Snippet: Agilent aCGH Wash Buffer 1 (Agilent, cat. no. 5188-5221) Agilent aCGH Wash Buffer 2 (Agilent, cat. no. 5188-5222) EQUIPMENT 0.22-μm syringe filter (Nalgene, cat. no. 190-2520) 20-μm mesh filter (Celltrics, Partec, cat. no. 04-004-2325) MoFlo High Performance Cell Sorter equipped with two water-cooled lasers (Coherent, Innova 300 series) 1.5-ml microfuge tubes Benchtop microfuge Heat block Thermal cycler Microcon YM-30, 30 kDa, sample reservoirs and tubes (Millipore, cat. no. 42422) NanoDrop 2000 Spectrophotometer (Thermo Scientific) Hybridization Gasket Slide Kit (5) for 1 microarray per slide format (Agilent, cat. no. G2534-60003) Hybridization Chamber Kit (Agilent, cat. no. G2534A) Blunt forceps (supplied with hybridization chamber) Human Genome CGH Microarray Kit 244A, consists of 5 microarrays (Agilent, cat. no. G4411B) DNA Microarray Hybridization Oven (Agilent, cat. no. G2545A) set to 65 °C, 20 rpm Hybridization Oven Rotator Rack (Agilent, cat. no. G2530-60029) Three glass troughs One slide rack Microarray Scanner (we used Agilent G2565CA but G2565BA can be used, Agilent) Slide holders supplied with scanner Feature Extraction 9.5 or 10.5 software and Agilent Scan Control software—both supplied with scanner DNA Analytics 4.0 (Agilent, cat. no. G4172AA) Freely available ‘R’ software Summit v3.1 (analysis software from Dako/Beckman Coulter) REAGENT SETUP Cell culture medium Supplement 500 ml of RMPI 1640 or DMEM with 15% (vol/vol) fetal calf serum and antibiotics.

Techniques: Incubation, Isolation, Staining, Fluorescence, Negative Control, Produced, Labeling, Microarray, Hybridization

Journal: Science Progress

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

doi: 10.1177/00368504251372111

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

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

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

Journal: Science Progress

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

doi: 10.1177/00368504251372111

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

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

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

Journal: Genes, chromosomes & cancer

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

doi: 10.1002/gcc.22426

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

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

Techniques: Expressing, Derivative Assay, Microarray, Transformation Assay

Journal: OncoTargets and Therapy

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

doi: 10.2147/ott.s67570

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

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

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

Journal: OncoTargets and Therapy

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

doi: 10.2147/ott.s67570

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

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

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

Journal: Translational oncology

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

doi: 10.1016/j.tranon.2020.100987

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

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

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

Journal: Translational oncology

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

doi: 10.1016/j.tranon.2020.100987

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

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

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

Journal: Translational oncology

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

doi: 10.1016/j.tranon.2020.100987

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

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

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

Journal: Translational oncology

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

doi: 10.1016/j.tranon.2020.100987

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

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

Techniques: Expressing, Control, In Situ, Immunohistochemistry

Journal: Translational oncology

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

doi: 10.1016/j.tranon.2020.100987

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

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

Techniques: Expressing, In Situ Hybridization, Immunohistochemistry