Journal: Diabetologia

Article Title: Human adipose microRNA-221 is upregulated in obesity and affects fat metabolism downstream of leptin and TNF-α

doi: 10.1007/s00125-013-2950-9

Figure Lengend Snippet: miR-221 targets the 3′ UTRs of ADIPOR1 and ETS1 and decreases their protein levels. ( a,b ) TargetScan conserved predicted miR-221 binding site on the 3′ UTRs of ADIPOR1 and ETS1 . ( c,d ) Quantification of dual-luciferase assay in HEK 293 cells co-transfected with miR-221 mimic (black bars) or control oligonucleotide (white bars) and ADIPOR1/2 ( c ) or ETS1 ( d ) 3′ UTR reporter plasmids. ( d ) Two overlapping fragments of the ETS1 3′-UTR were subcloned into separate vectors, indicated (A) and (B). Firefly/renilla luciferase ratio (luc ratio) was normalised to no-UTR controls. Error bars indicate SD ( n = 5). * p < 0.05 (two-tailed Mann–Whitney U test). ( e ) Quantification of QRT-PCR for ETS1 mRNA in human pre-adipocytes transfected with miR-221 mimic (black bars) or control oligonucleotide (white bars). Error bars indicate SD ( n = 3). ( f ) Immunoblots for ADIPOR1, ETS1 and β-actin in human pre-adipocytes transfected with miR-221 mimic (+) or control oligonucleotide. ( g ) Quantification of the immunoblots shown in ( f ). Background-subtracted mean signal ( n = 2) for ADIPOR1 and ETS1, normalised to the loading control (β-actin). Black bars, miR-221 mimic; white bars, control oligonucleotide

Article Snippet: For 3′ untranslated (UTR) reporter assays, JetPrime (Polyplus-transfection SA, Illkirch, France) was used to co-transfect HEK 293 cells (ATCC) with miRNA 3′ UTR target expression vectors for human ETS1 , ADIPOR1 , ADIPOR2 or control (Genecopoeia, Germantown, MD, USA), and human miR-221 mimic (Dharmacon, Lafayette, CO, USA) or scrambled control oligonucleotide, in 24-well plates.

Techniques: Binding Assay, Luciferase, Transfection, Control, Two Tailed Test, MANN-WHITNEY, Quantitative RT-PCR, Western Blot

Journal: Cell Host & Microbe

Article Title: Aspergillus fumigatus hijacks human p11 to redirect fungal-containing phagosomes to non-degradative pathway

doi: 10.1016/j.chom.2023.02.002

Figure Lengend Snippet:

Article Snippet: HCP216549-SG01-3 for generation of p11-KO cell line , GeneCopoiea , Cat# HCP216549-SG01-3.

Techniques: Virus, Recombinant, Cell Culture, Protease Inhibitor, Transfection, SYBR Green Assay, Mass Spectrometry, CyQUANT Assay, LDH Cytotoxicity Assay, cDNA Synthesis, Purification, Gene Expression, Construct, Control, Transformation Assay, Cloning, Plasmid Preparation, Expressing, Software, Imaging, Blocking Assay, Red Blood Cell Lysis, Membrane

Journal: PLoS ONE

Article Title: Downregulation of miR-205 Modulates Cell Susceptibility to Oxidative and Endoplasmic Reticulum Stresses in Renal Tubular Cells

doi: 10.1371/journal.pone.0041462

Figure Lengend Snippet: PCR primers used for detection of mRNAs.

Article Snippet: The EGLN2 3′-UTR miRNA target sequence expression clone in pEZX-MT01 vector with fLuc was purchased from GeneCopoeia (miTarget TM miRNA 3′-UTR Target Clone, Rockville, MD).

Techniques:

Journal: PLoS ONE

Article Title: Downregulation of miR-205 Modulates Cell Susceptibility to Oxidative and Endoplasmic Reticulum Stresses in Renal Tubular Cells

doi: 10.1371/journal.pone.0041462

Figure Lengend Snippet: (A) Quantitative real-time RT-PCR analysis revealed that the expression of EGLN2 was increased by miR-205 inhibition, but not significantly altered by overexpression of miR-205 compared to negative control transfected HK-2. No changes were seen in expression levels of EGLN1 (PHD2) or EGLN3 (PHD3). The data represent the means ± S.E. of triplicate analysis from three independent experiments. *P<0.05 by Dunnett’s multiple comparison test. (B) Western blot analysis showed that EGLN2 was also increased at protein level by miR-205 inhibition. The result of densitmetry represent the means ± S.E. of triplicate analysis from two independent experiments.**P<0.01 by standard t -test. (C) Quantitative real-time RT-PCR analysis revealed that the expression level of EGLN2 was increased in HK-2 exposed to hypoxia-reoxygenation or TUN, but these increases were supressed by miR-205 overexpression (▪) compared to negative control transfected HK-2 (□). *P<0.05 by Tukey’s multiple comparison test. (D) EGLN2 (transcript variant 1) contains the predicted binding site for miR-205 in its 3′-UTR. The predicted pairing of the mRNA target region (top) and miRNA (bottom) is as indicated, wherein solids line indicates hydrogen base pairing and the dotted line indicates Watson-Crick/wobble base pairing. The shaded sequence indicates the seed region of miR-205. (E) Mutant 3′-UTR sequence that abolished binding to miR-205. (F) HK-2 cells were transiently cotransfected with a luciferase reporters expressing EGLN2 3′-UTR or mutant EGLN2 3′-UTR miRNA target sequence and miR-205 mimic, miR-205 inhibitor, or negative control. At 24 hours posttransfection, firefly luciferase activities were measured and normalized with Renilla luciferase activities. Relative luciferase activity was increased by miR-205 inhibition when compared with control, while mutant EGLN2 3′-UTR had no effect on luciferase activity, indicating that miR-205 acts on the 3′-UTR of the EGLN2. Though transfection of HK-2 with EGLN2 3′-UTR vector along with miR-205 overexpression tended to decrease luciferase activity, this change was not sicnificant. The data represent the means ± S.E. of triplicate measurements from two independent experiments. *P<0.05 by Tukey’s multiple comparison test. (G) When transiently transfected HK-2 with EGLN2 3′-UTR vector were exposed to oxidative or ER stress, the relative luciferase activities were significantly increased, and these increases were canceled with mutant EGLN2 3′-UTR vectors. Furthermore, co-transfection of EGLN2 3′-UTR vector with miR-205 mimic partially restrained these stress-induced increases of luciferase activities. The data represent the means ± S.E. of triplicate measurements from two independent experiments. *P<0.05 by Tukey’s multiple comparison test.

Article Snippet: The EGLN2 3′-UTR miRNA target sequence expression clone in pEZX-MT01 vector with fLuc was purchased from GeneCopoeia (miTarget TM miRNA 3′-UTR Target Clone, Rockville, MD).

Techniques: Quantitative RT-PCR, Expressing, Inhibition, Over Expression, Negative Control, Transfection, Comparison, Western Blot, Variant Assay, Binding Assay, Sequencing, Mutagenesis, Luciferase, Activity Assay, Control, Plasmid Preparation, Cotransfection

Journal: PLoS ONE

Article Title: Downregulation of miR-205 Modulates Cell Susceptibility to Oxidative and Endoplasmic Reticulum Stresses in Renal Tubular Cells

doi: 10.1371/journal.pone.0041462

Figure Lengend Snippet: When cells are exposed to oxidative stress or ER stress, maladaptive down-regulation of miR-205 and subsequent up-regulation of EGLN2 occurs. ATF4 and HIF, which are negatively regulated by EGLN2, are supposed to contribute to the downstream suppression of anti-oxidant enzymes.

Article Snippet: The EGLN2 3′-UTR miRNA target sequence expression clone in pEZX-MT01 vector with fLuc was purchased from GeneCopoeia (miTarget TM miRNA 3′-UTR Target Clone, Rockville, MD).

Techniques: