microarray mrna expression analysis  (Illumina Inc)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis

doi: 10.1186/s13046-020-01814-5

Figure Lengend Snippet: CDK13 is upregulated in PCa tissues and the upregulation of CDK13 promoted proliferation in PCa cells. a , RT-qPCR detected CDK13 mRNA expression in 30 pairs of PCa and benign prostatic hyperplasia (BPH). * P < 0.05 vs. BPH. b , CDK13 protein expression was measured by Western blotting in 3 pairs of randomly selected BPH (B) and PCa (P) tissues. c , Hematoxylin and eosin (HE) staining of BPH and PCa tissues. d , Immunochemistry staining of CDK13 in BPH and PCa tissues. Scale bar = 50 mm. e , The expression levels of CDK13 mRNA in BPH and PCa tissues from the GSE13507 database ( P = 0.0007). f , The expression of CDK13 was examined by RT-qPCR (up panel) or Western blotting (down panel) in human normal prostate epithelial cells (RWPE-1) and PCa cell lines (LNCaP, PC3, 22RV1 and DU145). CDK13 expression was significantly increased in PC3 and 22RV1 cell lines. * P < 0.05 vs. RWPE-1. g and h , Cell viability was measured by MTS assay (G) and colony formation assays (H) in PC3 and 22RV1 cell lines after transfected with indicated vectors. * P < 0.05, and ** P < 0.01 vs. their respective empty vector. i , PC3 and 22RV1 cells were transfected with shCDK13 or control vector, cell apoptosis was detected by AnnexinV/PI flow cytometry. Right panel shows the apoptosis rate of three independent experiments. ** P < 0.001 vs. pLKO. pWPI is a control vector of oeCDK13 and pLKO is the control vector of shCDK13

Article Snippet: Microarray hybridization analysis of mRNA expression in 2 PCa samples and 2 BPH were performed according to the manufacturer’s protocol (Arraystar, Inc., Rockville, MD, USA).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Staining, MTS Assay, Transfection, Plasmid Preparation, Control, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis

doi: 10.1186/s13046-020-01814-5

Figure Lengend Snippet: CDK13 interacts with E2F5 in PCa cells. a , Co-immunoprecipitation coupled with mass spectrometry (CoIP-MS) was performed with an anti-CDK13 in PC3 cells to detect the proteins interacting with CDK13, which are shown on the right panel. b , Heat map showing the differential expression (fold changes) of mRNAs between PCa and BPH tissues. Red color indicates several genes that are known to be transcriptionally upregulated in PCa tissues. c , CoIP analysis was used to detect the interaction between CDK13 and E2F5, and β-actin was used as a negative control. d , in situ proximity ligation (PLA) analysis detected the interaction between CDK13 and E2F5. Red color indicates PLA-positive cells. e , Immunofluorescence staining was performed to detect the expression and location of CDK13 and E2F5 in PCa and BPH tissues. Scale bars = 100 μm. f , E2F5 mRNA level was determined by RT-qPCR in 30 pairs of PCa and normal prostate tissues. ** P < 0.01 vs. normal tissues. g , The expression levels of E2F5 mRNA in BPH and PCa tissues from the GSE13507 database ( P < 0.0001). h , E2F5 protein expression was detected by Western blotting in 3 pairs of randomly selected BPH (B) and PCa (P) tissues. i , Immunohistochemistry staining detected the E2F5 protein expression in PCa and BPH tissues. j and k , the correlation between CDK13 and E2F5 mRNA expression in the PCa tissues was analyzed by Pearson correlation analysis in our clinical data ( R = 0.4928, P = 0.0049) or several other data published in TCGA database (Yu: R = 0.3959, P = 0.0001; Wallace: R = 0.3752, P = 0.0015; Glinsky: R = 0.238, P = 0.0347)

Article Snippet: Microarray hybridization analysis of mRNA expression in 2 PCa samples and 2 BPH were performed according to the manufacturer’s protocol (Arraystar, Inc., Rockville, MD, USA).

Techniques: Immunoprecipitation, Mass Spectrometry, Quantitative Proteomics, Negative Control, In Situ, Ligation, Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis

doi: 10.1186/s13046-020-01814-5

Figure Lengend Snippet: Transcriptional activation of endogenous CDK13 upregulates E2F5 expression by promoting circCDK13 formation. a , PC3 cells were transfected with CDK13 sgRNA, oeCDK13 or their respective control vector, and then Western blot analysis detected the CDK13 and E2F5 expression. b , PC3 cells were treated as in (A), RT-qPCR detected E2F5 mRNA expression. c and d , PC3 cells were transfected with shCDK13 or pLKO or treated with CDK13 inhibitor THZ531, and then Western blot (C) and RT-qPCR (D) analysis detected the CDK13 and E2F5 expression. e and f , PC3 cells were transfected with CDK13 sgRNA or NC sgRNA and treated with actinomycin D (Act D). Western blotting (E) detected E2F5 protein level, and RT-qPCR (F) detected the expression of circRNAs (hsa_circ_0001699, hsa_circ_0079929, hsa_circ_0079933 and hsa_circ_0079939). * P < 0.05 vs. NC sgRNA+DMSO; # p <0.05 vs. CDK13 sgRNA+DMSO. g , PCR was used to detect hsa_circ_0079929 (termed circCDK13) in PCa tissues by using convergent or divergent primers. Divergent primers amplify circCDK13 in cDNA but not in genomic DNA (gDNA). GAPDH was used as linear control. h , RT-PCR amplified full-length circCDK13 in PC3 cells, and the amplified products were confirmed by agarose gel electrophoresis. i , Sanger sequencing confirmed head-to-tail splicing of circCDK13. j , RT-qPCR detected the expression of circCDK13 in PCa and BPH tissues. ** P < 0.01 vs. BPH. k , The correlation between circCDK13 and E2F5 mRNA expression in our clinical data was analyzed by Pearson correlation analysis ( R = 0.3976, P = 0.0296). l , PC3 and 22RV1 cells were transfected with si-circCDK13, si-linear CDK13 or si-Ctl. RT-qPCR detected circCDK13 and CDK13 mRNA expression. Bars are mean ± SEM of triplicate samples. * P < 0.05, ** P < 0.01 vs. si-Ctl. m , RT-qPCR examined the expression of CDK13 mRNA and circCDK13 in PC3 and 22RV1 cells transfected with circCDK13 or empty vector. * P < 0.05, ** P < 0.01 vs. empty vector. n , Western blotting detected the E2F5 expression in PC3 and 22RV1 cells transfected with si-circCDK13, circCDK13 or their respective control. o , PC3 and 22RV1 cells were transfected with circCDK13 and shE2F5 either alone or together, and then colony formation assay was performed to detect the cell proliferation. * P < 0.05 vs. pLKO+pWPI, # p <0.05 vs. circCDK13 + pLKO

Article Snippet: Microarray hybridization analysis of mRNA expression in 2 PCa samples and 2 BPH were performed according to the manufacturer’s protocol (Arraystar, Inc., Rockville, MD, USA).

Techniques: Activation Assay, Expressing, Transfection, Control, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Sequencing, Colony Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis

doi: 10.1186/s13046-020-01814-5

Figure Lengend Snippet: circCDK13 upregulates E2F5 protein level by sequestering miR-221-5p/449a a , RT-qPCR detected the expression of E2F5 mRNA in PC3 cells transfected with circCDK13, si-circCDK13 or their respective control. b , Venn diagram displaying potential microRNAs associated with circCDK13 sequence from three online target-prediction programs. c , circCDK13 or empty vector was transfected into PC3 cells, RT-qPCR detected the pulled down efficiency of circCDK13 by biotinylated probes against circCDK13. * P < 0.05, ** P < 0.01 vs. con probe or empty vector. d , RT-qPCR detected the level of indicated miRNAs pulled down by biotinylated probes against circCDK13. * P < 0.05, ** P < 0.01 vs. con probe. e , Biotin-labeled E2F5 3′-UTR RNA was transfected into PC3 cells followed by a biotin pull-down assay using Streptavidin-coupled Dynabeads. The miRNAs were extracted from the sedimented beads, and the relative levels of 11 candidate miRNAs were detected by RT-qPCR. * P < 0.05, ** P < 0.01 vs. control E2F5 3′-UTR. f , PC3 cells were co-transfected with miR-449a mimic, miR-212-5p mimic or both and circCDK13-directed luciferase reporter. Luciferase activity was measured by dual-luciferase reporter assays. * P < 0.05, ** P < 0.01 vs. miR-NC. g , RNA in situ hybridization detected the co-localization between miR-449a or miR-212-5p with circCDK13 (arrowheads) in PC3 cells co-transfected with circCDK13 expression vector and miR-449a or miR-212-5p mimic. Nuclei were counterstained with DAPI. Scale bars = 25 μm. h , PC3 cells were co-transfected with miR-449a, miR-212-5p or control mimic (miR-NC) and wild type or mutated (mut) E2F5 3’UTR-directed luciferase reporter. Luciferase activity was measured by dual-luciferase reporter assays. Graph bars are mean ± SEM of 3 independent experiments. * P < 0.05, ** P < 0.01 vs. miR-NC or E2F5 3’UTR mut. i , PC3 and 22RV1 cells were transfected with miR-212-5p, miR-449a mimic or both, and then Western blotting detected the CDK13 and E2F5 expression.

Article Snippet: Microarray hybridization analysis of mRNA expression in 2 PCa samples and 2 BPH were performed according to the manufacturer’s protocol (Arraystar, Inc., Rockville, MD, USA).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, Sequencing, Plasmid Preparation, Labeling, Pull Down Assay, Luciferase, Activity Assay, RNA In Situ Hybridization, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Hepatocyte specific HMGB1 deletion worsens the injury in liver ischemia/reperfusion: A role for intracellular HMGB1 in cellular protection

doi: 10.1002/hep.26976

Figure Lengend Snippet: (A) RT-PCR was used to determine mRNA levels of HMGB1 within isolated hepatocytes from control and HMGB1-HC-KO mice. (B) HMGB1 protein levels within isolated hepatocytes or non-parenchymal cells from control and HMGB1-HC-KO mice were assessed by Western blot analysis. Figure is representative of three experiments with similar results. (C) Immunofluorescent stain of HMGB1 within cultured hepatocytes from control and HMGB1-HC-KO mice (magnification ×400). Images are representative of three experiments with similar results. Green, HMGB1; blue, nuclei; red, F-actin. (D) Serum HMGB1 ELISA after 1h or 6h of reperfusion. *P<0.05 when compared against control. (E) Immunofluorescent stain of HMGB1 within from sections of normal liver and liver 6h after I/R in control and HMGB1-HC-KO mice (magnification ×400). Images are representative liver sections from six mice per group. Red, HMGB1; blue, nuclei; green, F-actin.

Article Snippet: University of Pittsburgh Genomics and Proteomics Core Laboratories performed mRNA microarray expression analysis using Illumina bead-array platform with Mouse Refseq8 BeadChip.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Control, Western Blot, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Hepatocyte specific HMGB1 deletion worsens the injury in liver ischemia/reperfusion: A role for intracellular HMGB1 in cellular protection

doi: 10.1002/hep.26976

Figure Lengend Snippet: (A) TNF-α, IL-6 and IL-1β mRNA expression was determined in non-parenchymal cells cultured overnight with media from hypoxic HMGB1 KO or control hepatocytes. Non-parenchymal cells were treated with TLR9 antagonist, ODN2088, or ODN control. Results are expressed as the relative increase of mRNA expression compared with PBS treatment. Data represent the mean ± SE and are representative of three experiments with similar results. *P < 0.05 compared to ODN control-treated HMGB1-HC-KO group. **P < 0.05 compared to ODN control-treated HMGB1 Control group. (B) Serum ALT levels in HMGB1-HC-KO or control mice after 6h of reperfusion that were treated with ODN2088 or ODN control. Data represent the mean ± SE (n = 6 mice per group). *P < 0.05 compared to ODN control-treated HMGB1-HC-KO group. **P < 0.05 compared to ODN control-treated HMGB1 Control group. (C) TNF-α, IL-6 and IL-1β mRNA expression was determined in non-parenchymal cells cultured overnight with media from hypoxic HMGB1 KO or control hepatocytes. Hepatocytes were pre-treated with the PARP-1 inhibitor, 3-AB or PJ-34, or negative control, PBS. Results are expressed as the relative increase of mRNA expression compared with PBS treatment. Data represent the mean ± SE and are representative of three experiments with similar results. *P < 0.05 compared to PBS-treated HMGB1-HC-KO group. **P < 0.05 compared to PBS-treated HMGB1 Control group. (D) Serum ALT levels in HMGB1-HC-KO or control mice after 6h of reperfusion that were treated with the PARP-1 inhibitor, 3-AB or PJ-34, or PBS. Data represent the mean ± SE (n = 6 mice per group). *P < 0.05 compared to PBS-treated HMGB1-HC-KO group. **P < 0.05 compared to PBS-treated HMGB1 Control group.

Article Snippet: University of Pittsburgh Genomics and Proteomics Core Laboratories performed mRNA microarray expression analysis using Illumina bead-array platform with Mouse Refseq8 BeadChip.

Techniques: Expressing, Cell Culture, Control, Negative Control

Journal: Nucleic Acids Research

Article Title: Identification of preferential target sites for human DNA methyltransferases

doi: 10.1093/nar/gkq774

Figure Lengend Snippet: Generation of cell lines stably overexpressing DNA methyltransferases (DNMTs). ( A ) Schematic structure of DNMT isoforms and expression vector constructs. The conserved PWWP and PHD-like domains, DNMT catalytic motifs (I, IV, VI, IX and X), and alternative splicing sites are indicated. The coding regions of DNMT 3B4 and DNMT 3B5 in red represent frame shift mutations generated by alternative splicing. The expression vectors encode N-terminal myc -tagged DNMT isoforms. ( B ) mRNA expression of exogenous DNMTs. mRNA expression of each DNMT isoform was assessed by real time RT-PCR with primers designed for the IRES region located on a bicistronic transcript and normalized to the expression of GAPDH. ( C ) Protein expression levels of exogenous DNMT isoforms by western blotting using a myc antibody. The expression level of DNMTs shown in the right panel was lower than those in the left panel, such that a 10-fold higher amount of protein was loaded in the left panel, as shown by the GAPDH control. Note the 10-fold higher amount of DNMT 3A1 in the left panel was compared with 3A1 (1/10) in the right panel.

Article Snippet: Cell line 1 was subjected to Illumina DNA methylation analysis and mRNA expression microarray analysis.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Construct, Alternative Splicing, Generated, Quantitative RT-PCR, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: Identification of preferential target sites for human DNA methyltransferases

doi: 10.1093/nar/gkq774

Figure Lengend Snippet: Changes in mRNA expression and histone modification by overexpression of DNMT3A1 and DNMT3B1. Number of downregulated (>2-fold) genes in DNMT3A1- and DNMT3B1-overexpressing cells. The number of downregulated genes in whole genome mRNA representing 8276 transcripts ( A ) and in the 808 genes studied for DNA methylation ( B ). ( C ) Changes in mRNA expression and histone modification. Real-time PCR to measure the mRNA expression of DNMT3A1 target genes (EYA4 and HOXA11) and DNMT3B1 target genes (IGF2AS and CDH11), and ChIP combined with real-time PCR for histone medications (H3K4me3, H3K27me3 and H3K9me3) in HEK 293T cells (white bar), DNMT3A1 cells (black bar) and DNMT3B1 cells (gray bar). Real-time RT-PCR and ChIP data represent the means ± SD. For mRNA expression, the primer efficiency for all four genes was ∼95%, and comparison of the fold changes of the standard curve was consistent with the ΔCt-calculated fold changes. ( t -test; * P < 0.05 and *** P < 0.001 versus HEK 293T cells).

Article Snippet: Cell line 1 was subjected to Illumina DNA methylation analysis and mRNA expression microarray analysis.

Techniques: Expressing, Modification, Over Expression, DNA Methylation Assay, Real-time Polymerase Chain Reaction, Medications, Quantitative RT-PCR, Comparison