Journal: International Journal of Genomics

Article Title: Tissue- and Cell Type-Specific Expression of the Long Noncoding RNA Klhl14-AS in Mouse

doi: 10.1155/2017/9769171

Figure Lengend Snippet: Klhl14-AS expression in adult mouse organs. Quantitative real-time RT-PCR was performed by amplifying a region shared by Klhl14-AS isoforms on total RNA from adult mouse organs. The data are reported as normalized by Abelson expression. Three replicates

Article Snippet: Total cDNA was generated with the SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen 18080051), according to the manufacturer's specifications.

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Genomics

Article Title: Tissue- and Cell Type-Specific Expression of the Long Noncoding RNA Klhl14-AS in Mouse

doi: 10.1155/2017/9769171

Figure Lengend Snippet: Klhl14-AS isoform expression in adult mouse tissues. Klhl14-AS transcripts were amplified by RT-PCR on total RNA from adult mouse organs. Three different PCR reactions were performed on the same template cDNA. Upper image: A and B isoforms were amplified

Article Snippet: Total cDNA was generated with the SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen 18080051), according to the manufacturer's specifications.

Techniques: Expressing, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

Journal: Cancer Cell International

Article Title: LncRNA HOXA-AS2 promotes glioblastoma carcinogenesis by targeting miR-885-5p/RBBP4 axis

doi: 10.1186/s12935-020-01690-1

Figure Lengend Snippet: HOXA-AS2 was the target gene of miR-885-5p. a starBase showed the binding site between HOXA-AS2 and miR-885-5p. b The potential binding site between HOXA-AS2 and miR-885-5p was identified using the dual-luciferase assay. Mimic, miR-885-5p mimic. WT-lnc, wild-type HOXA-AS2. MUT-lnc, mutant HOXA-AS2. c The interaction between HOXA-AS2 and miR-885-5p was identified using RIP analysis. d MiR-885-5p expression in glioblastoma tissues and non-tumor tissues was analyzed using qRT-PCR. N = 33. e The correlation analysis of miR-885-5p and HOXA-AS2. f MiR-885-5p expression in glioblastoma cell lines (U251 and U87) and normal human astrocytes cell line (NHA) was analyzed using qRT-PCR. The data were presented in the form of mean ± SD, and three independent experiments were performed. *P

Article Snippet: The SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Cat#: 11752050, Thermo Fisher Scientific, USA) was used to reverse-transcribe the lncRNAs and mRNAs into cDNA.

Techniques: Binding Assay, Luciferase, Mutagenesis, Expressing, Quantitative RT-PCR

Journal: Cancer Cell International

Article Title: LncRNA HOXA-AS2 promotes glioblastoma carcinogenesis by targeting miR-885-5p/RBBP4 axis

doi: 10.1186/s12935-020-01690-1

Figure Lengend Snippet: MiR-885-5p directly targeted RBBP4 by binding to its 3′UTR. a The potential binding site between miR-885-5p and RBBP4 was predicted using TargetScan Human 7.2. b The potential binding site between miR-885-5p and the 3′UTR of RBBP4 was demonstrated using the dual-luciferase assay. NC, negative control. Mimic, miR-885-5p mimic. c The interaction between RBBP4 and miR-885-5p was evaluated using the RNA pull-down assay. Bio-NC, biotinylated negative control. Bio-miR-885-5p, biotinylated miR-885-5p. d RBBP4 expression in glioblastoma tissues and normal tissues was detected by qRT-PCR. N = 33. e The correlation analysis of miR-885-5p and RBBP4. f RBBP4 expression in glioblastoma cell lines (U251 and U87) and normal human astrocytes cell line (NHA) were identified using qRT-PCR. g The expression of RBBP4 protein in glioblastoma cell lines (U251 and U87) and normal human astrocytes cell line (NHA) were detected with the western blot kit. h The expression of RBBP4 mRNA increased by miR-885-5p inhibitor in U87 and U251 cells. i The expression of RBBP4 protein increased by miR-885-5p inhibitor in U87 and U251 cells. NC, negative control. Inhibitor, miR-885-5p inhibitor. The cells in the blank group without any treatments. The data were presented in the form of mean ± SD, and three independent experiments were performed. *P

Article Snippet: The SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Cat#: 11752050, Thermo Fisher Scientific, USA) was used to reverse-transcribe the lncRNAs and mRNAs into cDNA.

Techniques: Binding Assay, Luciferase, Negative Control, Pull Down Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cancer Cell International

Article Title: LncRNA HOXA-AS2 promotes glioblastoma carcinogenesis by targeting miR-885-5p/RBBP4 axis

doi: 10.1186/s12935-020-01690-1

Figure Lengend Snippet: MiR-885-5p regulated by HOXA-AS2 could suppress the malignant phenotype of glioblastoma cells. a The transfection efficiency of si-HOXA-AS2 and miR-885-5p inhibitor was verified using qRT-PCR. b The viability ability of the transfected U87 and U251 cells was measured using CCK-8 assay. c The proliferation ability of the transfected U87 and U251 cells was measured using BrdU assay. d The adhesion ability of the transfected U87 and U251 cells was detected using cell adhesion assay. e The apoptosis ability of the transfected U87 and U251 cells was assessed using flow cytometry. The cells in the blank group without any treatments. The data were presented in the form of mean ± SD, and three independent experiments were performed. *P

Article Snippet: The SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Cat#: 11752050, Thermo Fisher Scientific, USA) was used to reverse-transcribe the lncRNAs and mRNAs into cDNA.

Techniques: Transfection, Quantitative RT-PCR, CCK-8 Assay, BrdU Staining, Cell Adhesion Assay, Flow Cytometry

Journal: Cancer Cell International

Article Title: LncRNA HOXA-AS2 promotes glioblastoma carcinogenesis by targeting miR-885-5p/RBBP4 axis

doi: 10.1186/s12935-020-01690-1

Figure Lengend Snippet: Si-HOXA-AS2 inhibited cell viability, cell proliferation and cell adhesion, but it induced cell apoptosis in glioblastoma cells. a HOXA-AS2 expression in glioblastoma tissues and non-tumor tissues was analyzed using qRT-PCR. N = 33. b HOXA-AS2 expression in glioblastoma cell lines (U251, U87, A172, SHG44 and SNB19) and normal human astrocytes cell line (NHA) was analyzed using qRT-PCR. c The intracellular distribution of HOXA-AS2 was identified using a subcellular fractionation location assay. d The transfection efficiency of si-HOXA-AS2 was verified in U87 and U251 cell lines by qRT-PCR. e The viability of the transfected U87 and U251 cells was measured after performing the CCK-8 assay. f The proliferation of the transfected U87 and U251 cells was measured using the BrdU assay. g The adhesion ability of the transfected U87 and U251 cells was evaluated using the cell adhesion assay. h The apoptosis rate of the transfected U87 and U251 cells was evaluated using flow cytometry. i The Twist, Slug, Vimentin, MMP-2 protein expression level of the transfected U87 and U251 cells was evaluated using western blot assay. NC, negative control. Si-LNC, si-HOXA-AS2. The cells in the blank group without any treatments. The data were presented in the form of mean ± SD, and three independent experiments were performed. *P

Article Snippet: The SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Cat#: 11752050, Thermo Fisher Scientific, USA) was used to reverse-transcribe the lncRNAs and mRNAs into cDNA.

Techniques: Expressing, Quantitative RT-PCR, Fractionation, Transfection, CCK-8 Assay, BrdU Staining, Cell Adhesion Assay, Flow Cytometry, Western Blot, Negative Control

Journal: Pharmaceuticals

Article Title: Eudebeiolide B Inhibits Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss by Regulating RANKL-Induced NF-κB, c-Fos and Calcium Signaling

doi: 10.3390/ph13120468

Figure Lengend Snippet: Eudebeiolide B inhibits RANKL-induced osteoclast differentiation and promotes osteoblast differentiation. ( A ) Structure of eudebeiolide B ( n = 3). ( B ) Cytotoxicity of eudebiolide B. Bone marrow macrophages (BMMs) were seeded and treated with indicated concentrations of eudebeiolide B in the presence of M-CSF (30 ng/mL) for 72 h. Cell viability was analyzed using the XTT assay ( n = 3). ( C ) BMMs were treated with RANKL (100 ng/mL) and M-CSF (30 ng/mL) after pretreatment with 1, 5, 10 or 30 μM of eudebeiolide B for 1 h. Cells were fixed and stained with TRAP staining solution. TRAP-positive multinucleated cells (TRAP + MNCs) with more than three nuclei were defined as osteoclasts and counted ( n = 3). ( D ) BMMs were seeded and treated with RANKL (50 ng/mL) and M-CSF (30 ng/mL) for 72 h. Cells were then treated with eudebeiolide B for 48 h. Bone resorption areas were measured using ImageJ ( n = 3). ( E ) MC3T3-E1 cells were seeded and incubated with differentiation media and eudebeiolide B for 7 days. Osteoblast differentiation was determined by ALP staining, and ALP activity was measured using the cell lysate ( n = 3). ( F ) MC3T3-E1 cells were seeded and incubated with differentiation media and eudebeiolide B for 21 days. Osteoblast differentiation was assessed by Alizarin red staining, and calcium accumulation was quantified with cetylpyridinium chloride solution ( n = 3). ( G ) MC3T3-E1 cells were treated with or without eudebeiolide B (10 μM) and cultured for 24, 48 and 72 h. The mRNA expression of Runx2, Osterix, OPG and RANKL was assessed by quantitative PCR ( n = 3). Values are expressed as the means ± S.D. of three individual experiments. * p

Article Snippet: The complementary DNA was synthesized from 1 µg/mL of total RNA using SuperScript III First-Strand Synthesis System for RT-PCR (Thermo Scientific).

Techniques: XTT Assay, Staining, Incubation, Activity Assay, Cell Culture, Expressing, Real-time Polymerase Chain Reaction

Journal: Pharmaceuticals

Article Title: Eudebeiolide B Inhibits Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss by Regulating RANKL-Induced NF-κB, c-Fos and Calcium Signaling

doi: 10.3390/ph13120468

Figure Lengend Snippet: Attenuation of RANKL-induced osteoclastogenesis-related transcription factor and gene expression by eudebeiolide B. ( A – C ) BMMs were pretreated with or without eudebeiolide B and stimulated with RANKL (100 ng/mL) for the indicated time. ( A ) Western blot analysis of c-Fos and NFATc1 protein was performed, and band densities of ( B ) c-Fos and ( C ) NFATc1 were quantified by ImageJ ( n = 3). ( D – G ) BMMs were cultured in the presence of RANKL (100 ng/mL) with or without eudebeiolide B for 48 h. ( D ) NFATc1, ( E ) cathepsin K, ( F ) MMP9 and ( G ) DC-STAMP mRNAs were analyzed by quantitative RT-PCR ( n = 3). All data are expressed as the means ± S.D. of three individual experiments. * p

Article Snippet: The complementary DNA was synthesized from 1 µg/mL of total RNA using SuperScript III First-Strand Synthesis System for RT-PCR (Thermo Scientific).

Techniques: Expressing, Western Blot, Cell Culture, Quantitative RT-PCR