Journal: Physiological Genomics

Article Title: Superoxide scavenging and Akt inhibition in myocardium ameliorate pressure overload-induced NF-?B activation and cardiac hypertrophy

doi: 10.1152/physiolgenomics.00202.2009

Figure Lengend Snippet: TAB-induced NF-κB activation is attenuated by AdCu/ZnSOD and AdDNAkt. Representative images at day 5 (A) and summary data (B) show the effects of AdLacZ, AdCu/ZnSOD, or AdDNAkt on NF-κB-driven luciferase activity (photon emission) over time in TAB mice compared with sham-operated mice. NF-κB activation was measured by in vivo bioluminescent imaging using Xenogen IVIS-200. In the pseudocolored scale, areas of high photon emission are displayed as red and areas of low photon emission are displayed as blue. Data are means ± SE (n = 4/group) expressed relative to sham-operated mice. *P < 0.05 vs. sham surgery; †P < 0.05 vs. TAB-AdLacZ. DNAkt, dominant-negative form of Akt.

Article Snippet: EMSA was performed with 6 μg of nuclear protein incubated with a γ- 32 P-labeled NF-κB consensus oligonucleotide probe 5′-AGTTGAGGGGACTTTCCCAGGC-3′ (Promega, Madison, WI) as described earlier ( 52 ).

Techniques: Activation Assay, Luciferase, Activity Assay, In Vivo, Imaging, Dominant Negative Mutation

Journal: World Journal of Gastroenterology : WJG

Article Title: 4-hydroxy-2, 3-nonenal activates activator protein-1 and mitogen-activated protein kinases in rat pancreatic stellate cells

doi: 10.3748/wjg.v10.i16.2344

Figure Lengend Snippet: PSCs were treated with IL-1β (at 2 ng/mL, lane 2) or HNE (at 5 μmol/L, lane 4) in serum-free medium for 1 h. Nuclear extracts were prepared and subjected to electro-phoretic mobility shift assay using AP-1 (panel A) or NF-κB (panel B) oligonucleotide probes. Arrows denote specific in-ducible complexes competitive with cold double-stranded oli-gonucleotide probes (lane 3). Lane 1: control (serum-free medium only). □: free probe.

Article Snippet: Double-stranded oligonucleotide probes for AP-1 and nuclear factor κB (NF-κB) were purchased from Promega (Madison WI).

Techniques: Mobility Shift

Journal: World Journal of Gastroenterology : WJG

Article Title: 4-hydroxy-2, 3-nonenal activates activator protein-1 and mitogen-activated protein kinases in rat pancreatic stellate cells

doi: 10.3748/wjg.v10.i16.2344

Figure Lengend Snippet: HNE increased AP-1, but not NF-κB, -dependent transcriptional activity. A: PSCs were transfected with the lu-ciferase vectors (2 X AP-1 or 2 X NF-κB) along with pRL-TK vector as an internal control. After 24 h, the transfected cells were treated with IL-1β (at 2 ng/mL) or HNE (at 1 μmol/L ). After another 24-h incubation, intracellular luciferase activities were determined. The data represent mean mean ± SD, calcu-lated from three independent experiments as fold induction compared with the activity observed in control (serum-free medium only, “Con”). bP < 0.01 vs control. RLU: relative light units. B: PSCs were treated with IL-1β (at 2 ng/mL) or HNE (at 1 μmol/L) in serum-free medium for the indicated times. Total cell lysates were prepared, and the level of IκB-α was deter-mined by Western blotting.

Article Snippet: Double-stranded oligonucleotide probes for AP-1 and nuclear factor κB (NF-κB) were purchased from Promega (Madison WI).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Incubation, Luciferase, Western Blot

Journal: Cell Death & Disease

Article Title: LncRNA RUNX1-IT1 which is downregulated by hypoxia-driven histone deacetylase 3 represses proliferation and cancer stem-like properties in hepatocellular carcinoma cells

doi: 10.1038/s41419-020-2274-x

Figure Lengend Snippet: a The real-time PCR data from our patient’s cohort revealed that RUNX1-IT1 was significantly downregulated in HCC tissues (T, n = 87) than that in normal tissues (NT, n = 87). *** P < 0.001 by Student’s t test. b The GEO data set (GSE54236) from R2: Genomics Analysis and Visualisation Platform ( http://r2.amc.nl ) indicated that the expression of RUNX1-IT1 was prominently lower in HCC tissues compared with normal liver tissues. *** P < 0.001 by Student’s t test. c The expressions of RUNX1-IT1 in human normal hepatocyte cell line L02 and HCC cell lines SK-HEP-1, PLC/PRF/5, MHCC-97L, MHCC-97H, Huh7, SMMC-7721, HepG2 and Hep3B were examined using qRT-PCR. n = three independent experiments, ** P < 0.01 or *** < 0.001 by Student’s t test versus LO2. d , e Kaplan–Meier survival curves of overall survival (OS) and disease-free survival (DFS) in our patients’ cohort. Patients were assigned into two subgroups according to the median expression of RUNX1-IT1. ** P < 0.01 by two-sided log-rank test.

Article Snippet: After that, the cells were incubated with prehybridization solution and hybridised using hybridisation solution and then incubated with Cy3-labelled RUNX1-IT1 oligonucleotide probe (obtained from RiboBio, Guangzhou, China) overnight.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: LncRNA RUNX1-IT1 which is downregulated by hypoxia-driven histone deacetylase 3 represses proliferation and cancer stem-like properties in hepatocellular carcinoma cells

doi: 10.1038/s41419-020-2274-x

Figure Lengend Snippet: a The expression level of RUNX1-IT1 in MHCC-97H cells was enhanced by pcDNA/RUNX1-IT1, and RUNX1-IT1 expression in HepG2 cells was reduced by sh-RUNX1-IT1 (shRNA#1, shRNA#2). n = three independent experiments, *** P < 0.001 by Student’s t test versus vector, or ** P < 0.01 by ANOVA versus shVector. pcDNA/RUNX1-IT1 repressed whereas RUNX1-IT1 shRNA promoted cell viability b , cell proliferation c , colony formation d and cell cycle progression e . n = three independent experiments, * P < 0.05, ** P < 0.01 or *** P < 0.001 by Student’s t test versus Vector or ANOVA versus shVector. f pcDNA/RUNX1-IT1 accelerated, whereas RUNXI-IT1 shRNA induced an inhibition of HCC cells apoptosis. n = three independent experiments, ** P < 0.01 by Student’s t test versus Vector or ANOVA versus shVector. Magnification of Edu is ×200, and scale bars = 50 μm. The scale bars of colony formation = 1 cm.

Article Snippet: After that, the cells were incubated with prehybridization solution and hybridised using hybridisation solution and then incubated with Cy3-labelled RUNX1-IT1 oligonucleotide probe (obtained from RiboBio, Guangzhou, China) overnight.

Techniques: Expressing, shRNA, Plasmid Preparation, Inhibition

Journal: Cell Death & Disease

Article Title: LncRNA RUNX1-IT1 which is downregulated by hypoxia-driven histone deacetylase 3 represses proliferation and cancer stem-like properties in hepatocellular carcinoma cells

doi: 10.1038/s41419-020-2274-x

Figure Lengend Snippet: a Transwell assays showed that RUNX1-IT1 restoration reduced invasive abilities of MHCC-97H cells, whereas RUNX1-IT1 depletion enhanced the invasion of HepG2 cells. Scale bars = 50 μm. n = three independent experiments, ** P < 0.01 by Student’s t test versus vector or ANOVA versus shVector. b Representative images of the tumorsphere formation assay were displayed after overexpression of RUNX1-IT1 in MHCC-97H cells and depletion of RUNX1-IT1 in HepG2 cells. The number of tumorspheres was counted and plotted, and the percentage of tumorspheres with diameters of 50–100 μm, 100–150 μm or >150 μm was calculated and plotted. Magnification is ×200, and scale bars = 50 μm. n = three independent experiments, ** P < 0.01 by Student’s t test versus Vector or ANOVA versus shVector. c The protein expression levels of EMT and CSC markers after overexpression of RUNX1-IT1 in MHCC-97H cells and depletion of RUNX1-IT1 in HepG2 cells were determined by western blot analysis. β-Actin was used as an internal control. n = three independent experiments, * P < 0.05, ** P < 0.01 by Student’s t test versus vector or ANOVA versus shVector. d Immunofluorescence analysis showed that RUNX1-IT1 restoration reduced CD44 expression in MHCC-97H cells, whereas RUNX1-IT1 depletion elevated the CCD44 expression in HepG2 cells. n = three independent experiments. Magnification is ×400, and scale bars = 20 μm.

Article Snippet: After that, the cells were incubated with prehybridization solution and hybridised using hybridisation solution and then incubated with Cy3-labelled RUNX1-IT1 oligonucleotide probe (obtained from RiboBio, Guangzhou, China) overnight.

Techniques: Plasmid Preparation, Tube Formation Assay, Over Expression, Expressing, Western Blot, Immunofluorescence

Journal: Cell Death & Disease

Article Title: LncRNA RUNX1-IT1 which is downregulated by hypoxia-driven histone deacetylase 3 represses proliferation and cancer stem-like properties in hepatocellular carcinoma cells

doi: 10.1038/s41419-020-2274-x

Figure Lengend Snippet: a Representative images of subcutaneous xenografts in nude mice implanted with MHCC-97H cells with Vector or overexpression RUNX1-IT1. ( n = 6 per group). b , c Xenografts weight (mg) and tumour sizes were monitored and undergone quantification analysis. n = 6, ** P < 0.01 by Student’s t test for tumour weight; ** P < 0.01 by repeated-measures ANOVA for tumour sizes. d Immunohistochemistry staining and the semi-quantification analysis of Ki-67, E-cadherin, Vimentin, CD44, Nanog and Sox2 in xenograft tissues from different groups. Magnification is ×400, the scale bar represents 20 μm. n = 6, * P < 0.05 or ** P < 0.01 by Student’s t test. e Representative images of orthotopic liver xenografts in nude mice implanted with MHCC-97H cells with Vector or overexpression RUNX1-IT1, representative hematoxylin and eosin–stained sections, and quantification analysis of tumour nodules in livers were displayed. Magnification is ×100, the scale bar represents 50 μm. n = 6, ** P < 0.01 by Student’s t test. f Representative hematoxylin and eosin–stained sections, and quantification analysis of metastasis nodules in lungs were shown. Magnification is ×100, the scale bar represents 50 μm. n = 6, ** P < 0.01 by Student’s t test.

Article Snippet: After that, the cells were incubated with prehybridization solution and hybridised using hybridisation solution and then incubated with Cy3-labelled RUNX1-IT1 oligonucleotide probe (obtained from RiboBio, Guangzhou, China) overnight.

Techniques: Plasmid Preparation, Over Expression, Immunohistochemistry, Staining

Journal: Cell Death & Disease

Article Title: LncRNA RUNX1-IT1 which is downregulated by hypoxia-driven histone deacetylase 3 represses proliferation and cancer stem-like properties in hepatocellular carcinoma cells

doi: 10.1038/s41419-020-2274-x

Figure Lengend Snippet: RNA FISH assays a and subcellular fractionation assays b indicated that RUNX1-IT1 was predominately located in the cytoplasm. Magnification is ×400, and scale bars = 20 μm. c By applying bioinformatics tools (MIRDB, and DINAN tools-LncBase Predicted v.2), we found that there were two putative binding sites between 3′-UTR of RUNX1-IT1-wild type (wt) and miR-632. RUNX1-IT1-mutant (mut) means mutation of binding sites in the 3′-UTR of RUNX1-IT1. d The expression of miR-632 in tumour tissues ( n = 87) was dramatically higher than that in adjacent non-tumour tissues ( n = 87). *** P < 0.001 by Student’s t test. e Pearson correlation analysis verified that there existed a negative association between miR-632 and RUNX1-IT1 in HCC tissues. f MiR-632 was elevated in HCC cell lines. n = three independent experiments, ** P < 0.01, *** P < 0.001 by Student’s t test versus L02. g Real-time PCR showed that miR-632 was negatively regulated by RUNX1-IT1. n = three independent experiments, *** P < 0.001 by Student’s t test versus vector or ANOVA versus shVector. h MiR-632 expression was greatly increased by miR-632 mimics, whereas significantly decreased by the inhibitors, and the expression of RUNX1-IT1 was negatively regulated by miR-632. n = three independent experiments, ** P < 0.01, *** P < 0.001 by ANOVA. i Luciferase reporter gene assays showed that miR-632 negatively regulated the luciferase activity of RUNX1-IT1-wt-3′-UTR, rather than of RUNX1-IT1-mut-3′-UTR. n = three independent experiments, ** P < 0.01, *** P < 0.001 by ANOVA. j The anti-Ago2 RIP assay with miR-632 mimics showed that both miR-632 and RUNX1-IT1 were enriched in Ago2 precipitate compared with IgG. n = three independent experiments. *** P < 0.001 by ANOVA. k RUNX1-IT1 was highly enriched in the sample pulled down by biotinylated wt miR-632 rather than mut miR-632. n = three independent experiments. *** P < 0.001 by ANOVA versus Bio-NC.

Article Snippet: After that, the cells were incubated with prehybridization solution and hybridised using hybridisation solution and then incubated with Cy3-labelled RUNX1-IT1 oligonucleotide probe (obtained from RiboBio, Guangzhou, China) overnight.

Techniques: Fractionation, Binding Assay, Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Luciferase, Activity Assay

Journal: Cell Death & Disease

Article Title: LncRNA RUNX1-IT1 which is downregulated by hypoxia-driven histone deacetylase 3 represses proliferation and cancer stem-like properties in hepatocellular carcinoma cells

doi: 10.1038/s41419-020-2274-x

Figure Lengend Snippet: Rescue experiments revealed that RUNX1-IT1 negatively regulated the miR-632 expression a , b , whereas positively regulated both the mRNA a , b and protein e , f expression of GSK-3β by RUNX1-IT1/miR-632/GSK-3β cascades. n = three independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001 by ANOVA. c , d Luciferase activity of TCF/LEF1 reporters was regulated by modulation of RUNX1-IT1/miR-632/ GSK-3β axis. n = three independent experiments. ** P < 0.01, *** P < 0.001 by ANOVA. e , f Western blot results from rescue experiments showed that RUNX1-IT1 negatively regulated the accumulation of cytoplasmic β-catenin and the accumulation of nuclear β-catenin, c-Myc expression and cyclin D1 level by RUNX1-IT1/miR-632/GSK-3β axis. n = three independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001 by ANOVA.

Article Snippet: After that, the cells were incubated with prehybridization solution and hybridised using hybridisation solution and then incubated with Cy3-labelled RUNX1-IT1 oligonucleotide probe (obtained from RiboBio, Guangzhou, China) overnight.

Techniques: Expressing, Luciferase, Activity Assay, Western Blot

Journal: Cell Death & Disease

Article Title: LncRNA RUNX1-IT1 which is downregulated by hypoxia-driven histone deacetylase 3 represses proliferation and cancer stem-like properties in hepatocellular carcinoma cells

doi: 10.1038/s41419-020-2274-x

Figure Lengend Snippet: a Pearson correlation analysis revealed that the expression levels of RUNX1-IT1 and HIF-1α were negatively correlated in 87 HCC samples, as measured by real-time PCR. b RUNX1-IT1 is downregulated, whereas miR-632 is upregulated under hypoxic culture conditions (1% O 2 , 5% CO 2 , 94% N 2 ) in HepG2 cells. n = three independent experiments. ** P < 0.01 by ANOVA. c Hypoxia increased HIF-1α and HDAC3 protein levels, whereas decreased GSK-3β and p-GSK-3β protein levels. n = three independent experiments. ** P < 0.01, *** P < 0.001 by ANOVA. d HepG2 cells were stimulated with varying concentrations of the histone deacetylase inhibitor trichostatin A (TSA) for 24 h, and RUNX1-IT1 was upregulated by the TSA. n = three independent experiments. * P < 0.05, ** P < 0.01 by ANOVA. e The repression of RUNX1-IT1 expression by hypoxia could be reversed by TSA. n = three independent experiments. * P < 0.05, ** P < 0.01 by ANOVA. f siRNA silencing HDAC3 arrested the downregulation of RUNX1-IT1 in hypoxia-induced HepG2 cells. n = three independent experiments. * P < 0.05 by Student’s t test. g Immunoblotting analysis revealed that the expression of hypoxia increased HIF-1α and HDAC3 protein levels, but decreased GSK-3β and p-GSK-3β protein levels. siRNA targeting HDAC3 was not influenced the expression of HIF-1α while erased the inhibition of GSK-3β and p-GSK-3β under hypoxic conditions. n = three independent experiments. ** P < 0.01, *** P < 0.001 by ANOVA. h qRT-PCR analysis displayed that siRNA targeting HIF-1α arrested the downregulation of RUNX1-IT1 in hypoxia-induced HepG2 cells. n = three independent experiments. ** P < 0.01 by Student’s t test. i Western blot analysis showed that siRNA targeting HIF-1α arrested the upregulation of HDAC3 protein level, and offset the downregulation of GSK-3β and p-GSK-3β protein levels in hypoxia-induced HepG2 cells. n = three independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001 by ANOVA.

Article Snippet: After that, the cells were incubated with prehybridization solution and hybridised using hybridisation solution and then incubated with Cy3-labelled RUNX1-IT1 oligonucleotide probe (obtained from RiboBio, Guangzhou, China) overnight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Histone Deacetylase Assay, Western Blot, Inhibition, Quantitative RT-PCR