Journal: The Journal of Biological Chemistry

Article Title: Downregulation of the endothelial histone demethylase JMJD3 is associated with neointimal hyperplasia of arteriovenous fistulas in kidney failure

doi: 10.1016/j.jbc.2022.101816

Figure Lengend Snippet: Effects of downregulated JMJD3 on EC survival and migration. A , ECs infected with lentivirus-mediated sgRNA targeting JMJD3 (sgJMJD3) or nonspecific sgRNA (Ctl) for 2 days at 90% confluence in a 12-well plate. Western blot detected the expressions of JMJD3, UTX, PCNA and methylation of H3K27. Pictures are representative of three independent experiments. B , quantification of Western blot analyses of the expressions of JMJD3 and PCNA. Statistical significance was analyzed by one-way ANOVA. C , ECs infected with lentivirus-mediated overexpression of JMJD3 (JMJD3OE) for 2 days at 90% confluence in a 12-well plate. ECs infected with lentivirus packaging with empty vector were used as negative control (Ctl). Western blot was used to detect the expressions of PCNA and methylation of H3K27. Photos are representative of three independent experiments. D , quantification of Western blot analyses in B . Statistical significance was analyzed by one-way ANOVA. E , ECs were cultured in 96-well plate and infected with lentivirus-mediated sgRNA targeting JMJD3 (sgJMJD3) or nonspecific sgRNA (Ctl). EC proliferation was determined by MTS during different time points. Data are presented as mean ± SD from three replicates. Statistical significance between groups was analyzed by two-way ANOVA. F and G , ECs were cultured in 12-well plate and infected with lentivirus-mediated sgRNA targeting JMJD3 (sgJMJD3) or nonspecific sgRNA (Ctl). Wound-healing assay was used to detect the EC migration ( F ). The rate of migration was measured by quantifying the total distance that the cells moved from the edge of the scratch toward the center of the scratch at 2 days or 4 days when compared with 0 days ( G ). EC, endothelial cell; JMJD3, Jumonji domain–containing protein-3; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PCNA, proliferating cell nuclear antigen; sgRNA, single-guide RNA; UTX, ubiquitously transcribed tetratricopeptide repeat, X chromosome.

Article Snippet: The ORF of mouse JMJD3 constructed into lentivirus vector was obtained from Addgene.

Techniques: Migration, Infection, Western Blot, Methylation, Over Expression, Plasmid Preparation, Negative Control, Cell Culture, Wound Healing Assay

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of the endothelial histone demethylase JMJD3 is associated with neointimal hyperplasia of arteriovenous fistulas in kidney failure

doi: 10.1016/j.jbc.2022.101816

Figure Lengend Snippet: JMJD3 mediates TGFβ1-induced EndMT. A , Western blot analysis of ECs overexpressing JMJD3 by lentivirus (JMJD3OE) for 2 days following treatment with 2 ng/ml TGFβ1 or solvent for another 5 days. ECs infected with lentivirus packaging with empty vector were used as negative control (Ctl). Photos are representative of three independent experiments. B , quantification of the expression of JMJD3 and H3K27me3 is shown as means ± SD. Statistical significance between two groups was analyzed by one-way ANOVA. C , quantification of the expression of VE-cadherin and α-SMA is shown as means ± SD. Statistical significance between groups was analyzed by two-way ANOVA. D and E , ECs were treated with 2 ng/ml TGFβ1 or solvent (Ctl) for 24 h. ChIP assays with rabbit anti-H3K27me3 or anti-H3K4me3 antibody were performed. Rabbit IgG was used as control. Real-time PCR amplified VE-cadherin promoter with specific primers, respectively. Quantitative PCR data were normalized to IgG negative control and displayed as fold enrichment and expressed as means ± SD. Statistical significance between groups was analyzed by one-way ANOVA. α-SMA, α-smooth muscle actin; ChIP, chromatin immunoprecipitation; EC, endothelial cell; EndMT, endothelial–mesenchymal transition; H3K27, histone H3 lysine 27; IgG, immunoglobulin G; JMJD3, Jumonji domain–containing protein-3; TGFβ1, transforming growth factor beta 1; VE-cadherin, vascular endothelial-cadherin.

Article Snippet: The ORF of mouse JMJD3 constructed into lentivirus vector was obtained from Addgene.

Techniques: Western Blot, Solvent, Infection, Plasmid Preparation, Negative Control, Expressing, Control, Real-time Polymerase Chain Reaction, Amplification, Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of the endothelial histone demethylase JMJD3 is associated with neointimal hyperplasia of arteriovenous fistulas in kidney failure

doi: 10.1016/j.jbc.2022.101816

Figure Lengend Snippet: Effects of JMJD3 knockdown in ECs on proliferation of VSMC. A , VSMC cells were plated on the bottom chamber of 96-well transwell cell culture system. ECs were cultured onto the top chamber of the transwell inserts and transduced with lentivirus to knockdown JMJD3 by sgRNA (sgJD) or overexpress JMJD3 (JDOE). ECs transduced with control lentivirus sgRNA (Ctl) or lentivirus packaging with empty vector (Con) were used as control. MTS assay was performed to detect cell proliferation after coculture for 72 h. Data are presented as mean ± SD for three replicates. B – D , VSMCs were cultured in 96-well plate or 12-well plate. SNP (ranged from 1 to 1000 μM) was added to treat VSMC for 48 h. VSMC proliferation was detected by MTS assay. Data are presented as mean ± SD from three replicates ( B ). PCNA expression was evaluated by Western blot ( C ) and density was quantified ( D ). E , ECs were cultured in 12-well plate and treated with lentivirus-mediated sgRNA targeting JMJD3 (sgJD) or overexpressed JMJD3 (JDOE) by lentivirus combined with or without L-NAME (300 μM). ECs transduced with control lentivirus sgRNA (Ctl) or lentivirus packaging with empty vector (Con) were used as control. Expression levels of JMJD3 and eNOS were detected by Western blot. Photos are representative of three independent experiments. F , quantification of Western blot analysis for eNOS is shown as mean ± SD. G , quantitative ChIP analysis of H3K27me3 at the eNOS promoter. ECs were transduced with lentivirus sgRNA targeting JMJD3 (sgJD) or control lentivirus nonspecific sgRNA (Ctl). ChIP assay was performed with rabbit anti-H3K27me3 antibodies. Rabbit IgG was used as control. Real-time PCR amplified eNOS promoter with specific primers. Quantitative PCR data were normalized to IgG negative control and displayed as fold enrichment and expressed as means ± SD. Statistical significance between groups was analyzed by two-way ANOVA. H , VSMCs were cocultured with ECs. VSMCs were plated on the bottom chamber of 12-well transwell cell culture system. ECs were cultured on the top chamber of transwell inserts and transduced with lentivirus to knockdown JMJD3 by sgRNA (sgJD) or overexpress JMJD3 (JDOE) with or without addition of SNP (100 μM) or L-NAME (300 μM). ECs transduced with control lentivirus sgRNA (Ctl) or lentivirus packaging with empty vector (Con) were used as control. After coculture for 48 h, VSMC lysates were used for detecting the expression levels of PCNA by Western blot. VSMC without EC coculture was also used as control (W/O). Photos are representative of three independent experiments. I , quantification of Western blot analysis for PCNA is shown as mean ± SD. All statistical significances between groups were analyzed by one-way ANOVA. ChIP, chromatin immunoprecipitation; EC, endothelial cell; eNOS, endothelial nitric oxide synthase; H3K27, histone H3 lysine 27; IgG, immunoglobulin G; JMJD3, Jumonji domain–containing protein-3; L-NAME, N (omega)-nitro- l -arginine methyl ester; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PCNA, proliferating cell nuclear antigen; sgRNA, single-guide RNA; SNP, sodium nitroprusside; VSMC, vascular smooth muscle cell.

Article Snippet: The ORF of mouse JMJD3 constructed into lentivirus vector was obtained from Addgene.

Techniques: Knockdown, Cell Culture, Transduction, Control, Plasmid Preparation, MTS Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Amplification, Negative Control, Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of the endothelial histone demethylase JMJD3 is associated with neointimal hyperplasia of arteriovenous fistulas in kidney failure

doi: 10.1016/j.jbc.2022.101816

Figure Lengend Snippet: JMJD3 is regulated by TGFβ1–Hes1 pathway in ECs. A , real-time PCR analysis of the mRNA level of JMJD3 after treatment of TGFβ1 or solvent (Ctl) for 24 h. Values of JMJD3 expression level were normalized to GAPDH . Data are presented as mean ± SD of three independent experiments. Statistical significance was measured by one-way ANOVA. B , ECs were stimulated with various concentration of TGFβ1 for 48 h. The expression of JMJD3 and methylation of H3K27 was determined by Western blot. C , quantification of Western blot analyses for JMJD3 is shown as mean ± SD. Statistical significance between groups was analyzed by one-way ANOVA. D , scheme of the mouse JMJD3 locus indicating two HES1-binding sites ( vertical arrows ), which were analyzed using JASPAR. Promoter region was used in the luciferase assay. E , Western blot of Hes1 in EC treatment with 2 ng/ml TGFβ1 or solvent for 24 h. Photos are representative of three independent experiments. F , quantification of Western blot analyses for Hes1 is shown as mean ± SD. Statistical significance between groups was analyzed by Mann–Whitney test. G , ECs were transduced with lentivirus overexpressing Hes1 (Hes OE) or empty vector (Ctl). ECs without transduction were also used as control (blank). Western blot was performed after 2 days of culture. Photos are representative of three independent experiments. H , quantification of Western blot analyses for Hes1, JMJD3, and H3K27me3 is shown as mean ± SD. Statistical significance between groups was analyzed by one-way ANOVA. I , ChIP assays with anti-Hes1 or anti-IgG antibody from ECs with 2 ng/ml TGFβ1 or solvent (Ctl) treatment for 24 h. JMJD3 promoter was amplified with specific primers by PCR with 2% input as template for 20 cycles or with immunoprecipitated DNA as template for 35 cycles. Photos are representative of three independent experiments. J , quantification of PCR products for JMJD3 promoter was analyzed by ImageJ and shown as mean ± SD. Statistical significance between groups was analyzed by two-way ANOVA. K , HEK293T cells were cotransfected with JMJD3 promoter and Hes1 recombinant plasmid or control plasmid. Luciferase activity of the JMJD3 promoter was compared in the presence and absence of Hes1. Data are presented as mean ± SD. Spots are presented as values of three replicates. Statistical significance between groups was analyzed by two-way ANOVA. ChIP, chromatin immunoprecipitation; EC, endothelial cell; HEK293T, human embryonic kidney 293T cell line; H3K27, histone H3 lysine 27; IgG, immunoglobulin G; JMJD3, Jumonji domain–containing protein-3; TGFβ1, transforming growth factor beta 1.

Article Snippet: The ORF of mouse JMJD3 constructed into lentivirus vector was obtained from Addgene.

Techniques: Real-time Polymerase Chain Reaction, Solvent, Expressing, Concentration Assay, Methylation, Western Blot, Binding Assay, Luciferase, MANN-WHITNEY, Transduction, Plasmid Preparation, Control, Amplification, Immunoprecipitation, Recombinant, Activity Assay, Chromatin Immunoprecipitation

Journal: BMC Molecular Biology

Article Title: Messenger RNA profile analysis deciphers new Esrrb responsive genes in prostate cancer cells

doi: 10.1186/s12867-015-0049-1

Figure Lengend Snippet: Characterization of Esrrb-expressing cancer cell line. Esrrb status of two independent replicates of stable transfected control DU145-pc3.1 and DU145-Esrrb cells are tested by a quantitative PCR b Western blot and c reverse transcriptase PCR. a Relative mRNA concentrations of Esrrb were measured by qPCR, Esrrb transcripts concentration were determined by standard curve method and Esrrb concentration were first normalized to the concentration of house keeping gene GAPDH, then normalized to Esrrb/GAPDH ratio of DU145-pc3.1 cells. b Total protein was extracted form HEK293, DU145-Esrrb and control DU145-pc3.1 cells. Protein concentration of Esrrb was determined by western blot using GAPDH as internal control. c RT-PCR was performed on total RNA extracted from HEK293, DU145-esrrb and control DU145-pc3.1 cells. Esrrb was expressed in DU145-Esrrb cells, while Esrrg is not expressed in either DU145-pc3.1 and DU145-Esrrb cells

Article Snippet: 70 % confluent DU145 cells were transfected with either pcDNA3.1-zeo (+)-Esrrb expression vector [ ], or control empty vector pcDNA3.1-zeo (+) (Promega, Madison, WI, USA).

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Concentration Assay, Protein Concentration, Reverse Transcription Polymerase Chain Reaction

Journal: BMC Molecular Biology

Article Title: Messenger RNA profile analysis deciphers new Esrrb responsive genes in prostate cancer cells

doi: 10.1186/s12867-015-0049-1

Figure Lengend Snippet: Esrrb-regulated mRNA validation. Left panel qPCR validation of Esrrb-regulated mRNAs. Expression values were firstly normalized to Gapdh and normalized ratios are further normalized to that of DU145-pc3.1. Error bars represent standard deviation. Student t test was performed for statistical analysis (*p < 0.05). Seven genes were differentially expressed in both RNA-seq and qPCR, 1 gene, TGFbeta, is not differentially expressed in either assay and serves as a negative control. Right panel RNA-Seq analysis result, fold change (FC) indicates the ratio of normalized read counts in DU145-Esrrb to that of DU145-pc3.1

Article Snippet: 70 % confluent DU145 cells were transfected with either pcDNA3.1-zeo (+)-Esrrb expression vector [ ], or control empty vector pcDNA3.1-zeo (+) (Promega, Madison, WI, USA).

Techniques: Expressing, Standard Deviation, RNA Sequencing Assay, Negative Control

Journal: BMC Molecular Biology

Article Title: Messenger RNA profile analysis deciphers new Esrrb responsive genes in prostate cancer cells

doi: 10.1186/s12867-015-0049-1

Figure Lengend Snippet: Transcriptome correlation and Esrrb altered mRNAs. a Transcriptome correlation analysis was performed using Spearman Ranking Correlation. Color represents the correlation coefficient. DY131 treatment to DU145-Esrrb cells results in the lowest correlation coefficient with DU145-pc3.1 cells. b Dot plot of Esrrb-induced gene expression alteration. Genes expressed at adequate level are tested for differential gene expression test. The plot was made by plotting the Log2FC (fold change) against the Log2 cpm (count-per-million) difference. Red color marks the genes that are significant differentially expressed (FDR < 0.05), and the blue lines marked the Log2FC cutoff value (Log2FC > 1 or Log2FC < −1). 67 genes passed both thresholds

Article Snippet: 70 % confluent DU145 cells were transfected with either pcDNA3.1-zeo (+)-Esrrb expression vector [ ], or control empty vector pcDNA3.1-zeo (+) (Promega, Madison, WI, USA).

Techniques: Expressing

Journal: Endocrinology

Article Title: Transcriptome Analyses of Female Somatotropes and Lactotropes Reveal Novel Regulators of Cell Identity in the Pituitary

doi: 10.1210/en.2018-00587

Figure Lengend Snippet: Impact of lineage-enriched transcription factors on expression of markers genes in Pit-1/Triple cells. Pit-1/Triple cells (5) were transfected with expression constructs encoding each noted transcription factor linked by an IRES to a GFP reporter open reading frame (see “Materials and Methods”). Transfected cells were isolated by GFP FACS and their RNA content was assayed for specific marker gene expression by qRT-PCR. (A) Nupr1. (B) Rxrg. (C) Nr4a2. (D) Pou4f1. In all assays, expression of the recombinant transcription factor was confirmed by targeted qRT-PCR (data not shown). Gene expression was normalized to an internal Gapdh control, and the fold change over empty vector controls was calculated using the ΔΔCt method (see “Materials and Methods”). Gapdh expression levels were stable across all assayed conditions, with no significant changes in Gapdh expression in any of the transfected samples. Error bars indicate 1 SD. n = 5 for all experiments. *P < 0.05, by t test.

Article Snippet: The cDNA of Nupr1 was amplified from mouse pituitary cDNA. cDNAs for the other studied transcription factors were obtained from OriGene or Addgene and subcloned into an internal ribosome entry site (IRES) GFP vector (Addgene plasmid no. 51406).

Techniques: Expressing, Transfection, Construct, Isolation, Marker, Gene Expression, Quantitative RT-PCR, Recombinant, Control, Plasmid Preparation

Journal: Endocrinology

Article Title: Transcriptome Analyses of Female Somatotropes and Lactotropes Reveal Novel Regulators of Cell Identity in the Pituitary

doi: 10.1210/en.2018-00587

Figure Lengend Snippet: NR4A2 acts in conjunction with POU1F1 at the Prl promoter to enhance Prl expression. (A) Schematic of the mPrl gene promoter indicating the relative positions of known POU1F1 binding sites [blue ovals (15)] as well as predicted binding sites for NR4A2 (red ovals) (identified by JASPAR). (B) NR4A2 ChIP. Pit-1/Triple cells were transfected with an expression vector encoding NR4A2, and chromatin isolated from GFP+ cells (as in Fig. 4) was assayed by NR4A2 ChIP. Levels of transcription factor occupancy at specific sites within the Prl promoter were quantified by qRT-PCR of immunoprecipitated samples. Parallel controls included quantitation of occupancy at the Gh promoter and assessment of binding in regions 500 bp upstream and downstream of the Prl promoter. The MyoD promoter served as a negative control in all studies. (C) Schematic of the mPrl gene promoter indicating the relative positioning of known POU1F1 binding sites (blue ovals) as well as predicted binding sites for POU4F1 (green ovals) (identified by JASPAR). (D) POU4F1 ChIP. Pit-1/Triple cells were transfected with an expression vector encoding POU4F1 and chromatin isolated from GFP+ cells [as in (B)] was assayed by ChIP. The Pou4f1 enhancer was assayed in this study as a positive control for the POU4F1 ChIP. (E) Nr4a2 ChIP performed in FACS-sorted mouse lactotropes. (F) Impact of NR4A2 on expression of markers genes in Pit-1/0 cells. Pit-1/0 cells were transfected with expression vectors encoding Pou1f1, Nr4a2, or both linked by IRES to GFP, and GFP+ cells were collected by FACS. qRT-PCR analysis was performed to measure changes in mRNA expression of somatotrope and lactotrope marker genes. n = 3 for all experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The cDNA of Nupr1 was amplified from mouse pituitary cDNA. cDNAs for the other studied transcription factors were obtained from OriGene or Addgene and subcloned into an internal ribosome entry site (IRES) GFP vector (Addgene plasmid no. 51406).

Techniques: Expressing, Binding Assay, Transfection, Plasmid Preparation, Isolation, Quantitative RT-PCR, Immunoprecipitation, Quantitation Assay, Negative Control, Positive Control, Marker

Journal: Endocrinology

Article Title: Transcriptome Analyses of Female Somatotropes and Lactotropes Reveal Novel Regulators of Cell Identity in the Pituitary

doi: 10.1210/en.2018-00587

Figure Lengend Snippet: ChIP of POU1F1 in Pit-1/Triple cells expressing Nr4a2 vs Pit-1/Triple cells that do not express Nr4a2. Pit-1/Triple cells, which do not express Nr4a2, were transfected with the same Nr4a2-IRES-GFP plasmid used in assays presented in Fig. 4C, and control cells were transfected with the empty IRES-GFP vector. GFP+ cells were sorted by FACS, chromatin was isolated, and ChIP was performed using an antibody that recognizes POU1F1. Following immunoprecipitation, qRT-PCR was used to measure the relative levels of POU1F1 binding in Pit-1/Triple cells that had been transfected with Nr4a2 plasmid (+Nr4a2), as well as those transfected with empty vector. Immunoprecipitations using IgG served as a negative control. *P < 0.05.

Article Snippet: The cDNA of Nupr1 was amplified from mouse pituitary cDNA. cDNAs for the other studied transcription factors were obtained from OriGene or Addgene and subcloned into an internal ribosome entry site (IRES) GFP vector (Addgene plasmid no. 51406).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Isolation, Immunoprecipitation, Quantitative RT-PCR, Binding Assay, Negative Control

Journal: Endocrinology

Article Title: Transcriptome Analyses of Female Somatotropes and Lactotropes Reveal Novel Regulators of Cell Identity in the Pituitary

doi: 10.1210/en.2018-00587

Figure Lengend Snippet: Immunoprecipitation of H3K27-acetylated (H3K27ac) chromatin in Pit-1/0 cells expressing Nr4a2 and/or Pou1f1. Pit-1/0 cells, which express neither Nr4a2 nor significant levels of Pou1f1, were transfected with Nr4a2-IRES-GFP, Pou1f1-IRES-GFP, or a 1:1 mixture of both expression vectors, and control cells were transfected with an empty IRES-GFP vector. Cells transfected with the 1:1 mixture of the Pou1f1 and Nr4a2 expression vectors express lower levels of each protein due to the concentration of each expression vector being halved during transfection to keep the total amount of DNA constant in each transfection. GFP+ cells were isolated by FACS and chromatin was isolated. ChIP was performed using an antibody that recognizes H3K27ac. Following ChIP, qRT-PCR was performed to assay the levels of H327 acetylation at the Gh promoter, the Prl promoter, and the MyoD promoter as a negative control. IgG controls (data not shown) were also included in this assay for all samples. In all cases, ChIP performed with IgG yielded <1% of input. *P < 0.05.

Article Snippet: The cDNA of Nupr1 was amplified from mouse pituitary cDNA. cDNAs for the other studied transcription factors were obtained from OriGene or Addgene and subcloned into an internal ribosome entry site (IRES) GFP vector (Addgene plasmid no. 51406).

Techniques: Immunoprecipitation, Expressing, Transfection, Control, Plasmid Preparation, Concentration Assay, Isolation, Quantitative RT-PCR, Negative Control

Journal: Cancer Science

Article Title: MicroRNA‐493‐5p‐mediated repression of the MYCN oncogene inhibits hepatic cancer cell growth and invasion

doi: 10.1111/cas.14292

Figure Lengend Snippet: Measurement of microRNA (miR)‐493‐5p and MYCN expression levels in clinical samples from hepatocellular carcinoma (HCC) patients. A,B, Relative expression levels of (A) miR‐493‐5p and (B) MYCN in clinical samples. Patients showing moderate to advanced hepatic tumors (length > 2.5 cm) were selected for the study. The box plots illustrate differential gene expression in 13 primary HCC tumors (T) compared with the corresponding nontumor tissues (NT). Mann–Whitney U test was used to calculate P values. C, Scatter plots of Spearman’s correlation coefficient analysis between miR‐493‐5p and MYCN relative expression, measured by real‐time quantitative PCR in all clinical samples (T and NT, N = 26). Red and blue plots show T and NT tissues, respectively

Article Snippet: For MYCN rescue experiments, the cells were incubated with 1.5 µg MYCN expression vector (plasmid #74163; Addgene) following the experimental procedure described above.

Techniques: Expressing, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: Cancer Science

Article Title: MicroRNA‐493‐5p‐mediated repression of the MYCN oncogene inhibits hepatic cancer cell growth and invasion

doi: 10.1111/cas.14292

Figure Lengend Snippet: Effect of MYCN knockdown and rescue on hepatic cancer cell growth and invasion. A, Hep3B cell growth assessment after MYCN silencing. Two distinct siRNAs were used to target MYCN (siMYCN_A and siMYCN_B). Scrambled siRNA was used as a negative control (siCtrl). Number of cells was estimated at the indicated times using a cell viability assay. B, Invasive abilities of Hep3B cells after MYCN knockdown. Cells that migrated through the Matrigel‐coated membrane were counted after 72 h. Photographs are representative of cell invasion for each condition. C, Cell viability. D, Invasion assay after MYCN and microRNA (miR)‐493‐5p overexpression in Hep3B cells (rescue experiment). MYCN expression vector did not contain MYCN mRNA 3′‐UTR. Cell viability was measured after 4 d. Validation of MYCN expression vector compared with the mock is shown in Figure . Data depicted in the figure show the mean ± SD. Significant differences were evaluated with a t test (n = 3). * P < .05, ** P < .01, *** P < .001

Article Snippet: For MYCN rescue experiments, the cells were incubated with 1.5 µg MYCN expression vector (plasmid #74163; Addgene) following the experimental procedure described above.

Techniques: Negative Control, Viability Assay, Invasion Assay, Over Expression, Expressing, Plasmid Preparation

Journal: Molecular Oncology

Article Title: Long noncoding RNA SNHG12 induces proliferation, migration, epithelial–mesenchymal transition, and stemness of esophageal squamous cell carcinoma cells via post‐transcriptional regulation of BMI1 and CTNNB1

doi: 10.1002/1878-0261.12683

Figure Lengend Snippet: SNHG12 upregulation promotes cell proliferation, migration, and EMT as well as cell stemness in ESCC. (A) qRT‐PCR confirmed SNHG12 overexpression by pcDNA3.1/SNHG12 ( n = 5; Student’s t ‐test). (B‐C) Colony formation and EdU (bar value = 100 μm) assays detected ESCC cell proliferation when cells were transfected with pcDNA3.1/SNHG13 ( n = 5; Student’s t ‐test). (D) Transwell assay measured ESCC cell migration ability when overexpressing SNHG12 (bar value = 100 μm; n = 5; Student’s t ‐test). (E) Western blot assay tested the expression of EMT‐related proteins (E‐cadherin, N‐cadherin, and vimentin; n = 5). (F) Sphere formation assay detected the ESCC cell sphere formation efficiency (bar value = 100 μm; n = 5; Student’s t ‐test). (G) Western blot assay measured the protein expression of stem cell markers (SOX2, SOX4, OCT4, and Nanog; n = 5). Results were all exhibited as the mean ± standard deviation (SD) and taken from more than three independent experiments. ** P < 0.01

Article Snippet: Besides, the pcDNA3.1/SNHG12, pcDNA3.1/BMI1, pcDNA3.1/IGF2BP2, pcDNA3.1/IGF2BP3, pcDNA3.1/SOX2, pcDNA3.1/SOX4, and their relative control pcDNA3.1 vectors were all procured from GeneChem (Shanghai, China).

Techniques: Migration, Quantitative RT-PCR, Over Expression, Transfection, Transwell Assay, Western Blot, Expressing, Tube Formation Assay, Standard Deviation