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: iScience

Article Title: Merkel cell polyomavirus T-antigens regulate DICER1 mRNA stability and translation through HSC70.

doi: 10.1016/j.isci.2021.103264

Figure Lengend Snippet: Figure 3. DICER1 mRNA is present in the LT-HSC70 complex and directly associated with HSC70 (A–C) Evaluation of DICER1 mRNA in LT or His-HSC70 immunoprecipitation (IP). RNA immmunoprecipitation was performed on (A) WaGa cells or (B) HEK293 cells transfected with LTco, LTcoD44N or pCTR, using LT antibody, as well as on (C) HEK293 cells co-transfected with His-HSC70 together with LTco or

Article Snippet: sTco (pcDNA6 MCV sTco) Shuda et al. (2011) Addgene Cat #40201 RRID: Addgene_40201 LT339 (pcDNA6 MCV LT339 V5.4) Shuda et al. (2008) Addgene Cat#28193 RRID: Addgene_28193 LT339D This paper N/A LT339E216K This paper N/A His-HSC70 (pcDNA5/FRT/TO HIS HSPA8) Hageman and Kampinga (2009) Addgene Cat#19541 RRID: Addgene_19541 shTA (shRNA targeting LT and sT) Xie et al. (2014) N/A shsT (shRNA targeting sT) Xie et al. (2014) N/A DICER1 30UTR (pIS1 DICER1 long UTR) Mayr and Bartel (2009) Addgene Cat#21649 RRID: Addgene_21649 DICER1 30UTR Mutsub This paper N/A DICER1 30UTR MutD This paper N/A AREMUT This paper N/A AREWT This paper N/A DICER1 CDSWT Paulsson et al. (2020) N/A DICER1 CDSD This paper N/A Software and algorithms R version 4.0.5 https://www.R-project.org/ N/A; RRID:SCR_001905 Rstudio http://www.rstudio.com/ N/A; RRID:SCR_000432 Rstatix https://github.com/kassambara/rstatix N/A; RRID:SCR_021240 tidyverse https://www.tidyverse.org/ N/A; RRID:SCR_019186 Prism GraphPad 8.0 GraphPad Software, La Jolla California USA N/A; RRID:SCR_002798

Techniques: Immunoprecipitation, Transfection

Journal: iScience

Article Title: Merkel cell polyomavirus T-antigens regulate DICER1 mRNA stability and translation through HSC70.

doi: 10.1016/j.isci.2021.103264

Figure Lengend Snippet: Figure 5. The nonameric ARE in the CDS partly contributes to LT-induced DICER1 expression (A) Illustration of plasmids containing the full-length CDS of DICER1 (CDSWT) or an in-frame deletion of aa1889-1891, which corresponds to the nonameric ARE (CDSD). (B) Western blot analysis of NoDice 2-20 cells co-transfected with DICER1 CDSWT and LTco, LTcoD44N, or pCTR. (C) The effect of LTco on exogenous DICER1 expression from DICER1 CDSWT and CDSD was evaluated in NoDice 2-20 cells using western blotting.

Article Snippet: sTco (pcDNA6 MCV sTco) Shuda et al. (2011) Addgene Cat #40201 RRID: Addgene_40201 LT339 (pcDNA6 MCV LT339 V5.4) Shuda et al. (2008) Addgene Cat#28193 RRID: Addgene_28193 LT339D This paper N/A LT339E216K This paper N/A His-HSC70 (pcDNA5/FRT/TO HIS HSPA8) Hageman and Kampinga (2009) Addgene Cat#19541 RRID: Addgene_19541 shTA (shRNA targeting LT and sT) Xie et al. (2014) N/A shsT (shRNA targeting sT) Xie et al. (2014) N/A DICER1 30UTR (pIS1 DICER1 long UTR) Mayr and Bartel (2009) Addgene Cat#21649 RRID: Addgene_21649 DICER1 30UTR Mutsub This paper N/A DICER1 30UTR MutD This paper N/A AREMUT This paper N/A AREWT This paper N/A DICER1 CDSWT Paulsson et al. (2020) N/A DICER1 CDSD This paper N/A Software and algorithms R version 4.0.5 https://www.R-project.org/ N/A; RRID:SCR_001905 Rstudio http://www.rstudio.com/ N/A; RRID:SCR_000432 Rstatix https://github.com/kassambara/rstatix N/A; RRID:SCR_021240 tidyverse https://www.tidyverse.org/ N/A; RRID:SCR_019186 Prism GraphPad 8.0 GraphPad Software, La Jolla California USA N/A; RRID:SCR_002798

Techniques: Expressing, Western Blot, Transfection

Journal: iScience

Article Title: Merkel cell polyomavirus T-antigens regulate DICER1 mRNA stability and translation through HSC70.

doi: 10.1016/j.isci.2021.103264

Figure Lengend Snippet: Figure 6. AUF1 is not involved in DICER1 regulation in MCC (A) Western blot analysis of DICER1 expression upon AUF1 silencing in MCPyV+ (WaGa and MKL1) and MCPyV (MCC26) cell lines. Two different siRNAs targeting AUF1 (siAUF1#1 and siAUF#2) were used. siCTR was applied as a negative control. (B) Quantification of DICER1 expression in Figure 6A. DICER1 expression was normalized to GAPDH and then siCTR. Error bars, SEM. Each biological replicate is presented as a circle. (C) RT-qPCR analysis of DICER1 expression in WaGa cells transfected with siCTR or siAUF1 after actinomycin D (Act. D) treatment. This experiment was conducted in parallel to the HSC70 silencing experiments described in Figure 2I. p value was assessed by two-way ANOVA. ns, not significant. (D) Western blot analysis of HSC70 and AUF1 in nuclear and cytoplasmic fractions of MCPyV+ cell lines. a-TUBULIN and HISTONE3 were used as cytoplasmic and nuclear markers, respectively. (E) Western blot analysis of HSC70 and AUF1 in cytosolic and nuclear compartments of MCC26 cells transfected with LT339 or pCTR. Lamin A/C was used as nuclear or nuclear membrane marker, whereas GAPDH was used as cytoplasmic marker. Red arrows indicate trLT of LT339.

Article Snippet: sTco (pcDNA6 MCV sTco) Shuda et al. (2011) Addgene Cat #40201 RRID: Addgene_40201 LT339 (pcDNA6 MCV LT339 V5.4) Shuda et al. (2008) Addgene Cat#28193 RRID: Addgene_28193 LT339D This paper N/A LT339E216K This paper N/A His-HSC70 (pcDNA5/FRT/TO HIS HSPA8) Hageman and Kampinga (2009) Addgene Cat#19541 RRID: Addgene_19541 shTA (shRNA targeting LT and sT) Xie et al. (2014) N/A shsT (shRNA targeting sT) Xie et al. (2014) N/A DICER1 30UTR (pIS1 DICER1 long UTR) Mayr and Bartel (2009) Addgene Cat#21649 RRID: Addgene_21649 DICER1 30UTR Mutsub This paper N/A DICER1 30UTR MutD This paper N/A AREMUT This paper N/A AREWT This paper N/A DICER1 CDSWT Paulsson et al. (2020) N/A DICER1 CDSD This paper N/A Software and algorithms R version 4.0.5 https://www.R-project.org/ N/A; RRID:SCR_001905 Rstudio http://www.rstudio.com/ N/A; RRID:SCR_000432 Rstatix https://github.com/kassambara/rstatix N/A; RRID:SCR_021240 tidyverse https://www.tidyverse.org/ N/A; RRID:SCR_019186 Prism GraphPad 8.0 GraphPad Software, La Jolla California USA N/A; RRID:SCR_002798

Techniques: Western Blot, Expressing, Negative Control, Quantitative RT-PCR, Transfection, Membrane, Marker

Journal: Diabetes

Article Title: miRNA-93 Inhibits GLUT4 and Is Overexpressed in Adipose Tissue of Polycystic Ovary Syndrome Patients and Women With Insulin Resistance

doi: 10.2337/db12-0963

Figure Lengend Snippet: GLUT4 expression in adipocytes of PCOS patients. A : GLUT4 mRNA expression was significantly reduced in primary subcutaneous (sc) adipocyte culture derived from PCOS patients ( n = 21) compared with matched control (CRL) subjects ( n = 20). B : GLUT4 protein was also reduced in PCOS. C : Taking IR into account, significant differences are observed in GLUT4 expression between IR and non-IR groups in primary adipocyte culture. D : In whole AT, GLUT4 is significantly lower in PCOS and further reduced if the patient demonstrated IR. E : In ATs from all patients studied, GLUT4 gene expression was negatively correlated to HOMA-IR ( r = −0.57, n = 25, P < 0.002). All data are shown as means ± SEM (error bars). * P < 0.05; ** P < 0.01.

Article Snippet: Primers for mature miRNAs and mouse Actb and Glut4 were purchased from OriGene (Rockville, MD). miR-103 was used as an internal control ( ).

Techniques: Expressing, Derivative Assay

Journal: Diabetes

Article Title: miRNA-93 Inhibits GLUT4 and Is Overexpressed in Adipose Tissue of Polycystic Ovary Syndrome Patients and Women With Insulin Resistance

doi: 10.2337/db12-0963

Figure Lengend Snippet: miR-93 represses GLUT4 in human adipocytes. Differentiated human adipocytes were transfected with miR-93 plasmid (2 μg) or empty vector. A : GLUT4 protein content was assessed in three independent experiments ( P < 0.03). RT-qPCR assessed GLUT4 gene expression ( B ) and miR-93 gene expression ( C ) in human differentiated adipocytes transfected with miR-93 ( P < 0.01). Results were obtained from three independent experiments. D and E : Protein expression of GLUT4 (red) was also assessed by immunofluorescence microscopy; scale bars are 300 μm.

Article Snippet: Primers for mature miRNAs and mouse Actb and Glut4 were purchased from OriGene (Rockville, MD). miR-103 was used as an internal control ( ).

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Immunofluorescence, Microscopy

Journal: Diabetes

Article Title: miRNA-93 Inhibits GLUT4 and Is Overexpressed in Adipose Tissue of Polycystic Ovary Syndrome Patients and Women With Insulin Resistance

doi: 10.2337/db12-0963

Figure Lengend Snippet: miR-93 targets Glut4 in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were transfected with miR-93 plasmid (2 μg) or empty vector. 3T3-L1 adipocytes with induced miR-93 ( A ) have decreased Glut4 gene expression ( B ). C and D : Comparison of Glut4 (red) in 3T3-L1 adipocytes transfected with an GFP-miR-93 vector or empty vector (green) and expression of Glut4 (red); scale bars are 50 μm. E : Luciferase Glut4 3′UTR plasmid was cotransfected with either miR-93 or empty vector. Results were obtained from three independent experiments. F and G : miR-93 inhibition increases Glut4 gene expression in 3T3-L1 adipocytes.

Article Snippet: Primers for mature miRNAs and mouse Actb and Glut4 were purchased from OriGene (Rockville, MD). miR-103 was used as an internal control ( ).

Techniques: Transfection, Plasmid Preparation, Expressing, Luciferase, Inhibition

Journal: Diabetes

Article Title: miRNA-93 Inhibits GLUT4 and Is Overexpressed in Adipose Tissue of Polycystic Ovary Syndrome Patients and Women With Insulin Resistance

doi: 10.2337/db12-0963

Figure Lengend Snippet: miR-93 is overexpressed in women with PCOS and associated with reduced GLUT4 and increased HOMA-IR. A : Observing miR-93 in the entire patient population, miR-93 is significantly increased in all PCOS ATs, as well as in control (CRL) subjects with IR (** P < 0.01 vs. control subjects). B : miR-93 is negatively correlated to GLUT4 expression in the AT from subjects studied ( r = −0.54, n = 25, P < 0.005). C : miR-93 is correlated with HOMA-IR in the AT from subjects studied ( r = 0.67, n = 25, P < 0.0002).

Article Snippet: Primers for mature miRNAs and mouse Actb and Glut4 were purchased from OriGene (Rockville, MD). miR-103 was used as an internal control ( ).

Techniques: Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: A lentiviral sponge for miR-101 regulates RanBP9 expression and amyloid precursor protein metabolism in hippocampal neurons

doi: 10.3389/fncel.2014.00037

Figure Lengend Snippet: The conserved miR-101 target site at the 507–513 bp region of the RanBP9 3′UTR is necessary for RanBP9 post-transcriptional regulation. (A) Ribonucleotide sequences of the conserved putative miR-101 RE within the RanBP9 3′UTR paired with the mature miR-101 sequence. (B) Schematic representation of the luciferase constructs used in this study. The miR-101 RE within the RanBP9 3′-UTR is underlined. In the mutant construct, the nucleotides at positions 4 and 5 of the miR-101 RE were mutated, as indicated in bold font. (C) SH-SY5Y neuroblastoma cells were transfected independently with the SC control plasmid or with each of two SC-RanBP9 luciferase reporter genes together with either miR-101 or a control microRNA (cel-miR-67) (100 nM). At 24 h post-transfection, luciferase activity was determined. Results are presented as the normalized activity of miR-101-transfected cells relative to that of cells transfected with cel-miR-67. Data are presented as the means ± the SE from three or four independent experiments ( ** p < 0.01, Student's t -test).

Article Snippet: The SC-RANBP9 3′UTR was purchased from Origene (SC210772).

Techniques: Sequencing, Luciferase, Construct, Mutagenesis, Transfection, Plasmid Preparation, Activity Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: A lentiviral sponge for miR-101 regulates RanBP9 expression and amyloid precursor protein metabolism in hippocampal neurons

doi: 10.3389/fncel.2014.00037

Figure Lengend Snippet: Inhibition of miR-101 in neurons increases levels of endogenous RanBP9, full-length APP, and its secreted product, sAPPβ . (A) Representative Western blot analysis of protein extracts from lentiviral vector-transduced hippocampal neurons at 7 days post-infection with an antibody recognizing RanBP9, an antibody recognizing full-length APP (4G8), or an antibody recognizing GAPDH. Full blot for RanBP9 with multiple samples is shown in Figure . (B) The intensities of the bands were quantified by densitometry. The results obtained with the 4G8 antibody or the RanBP9 antibody were normalized to those obtained with the GAPDH antibody and expressed as arbitrary optical density (OD) units. The band intensities for miR-101 sponge-containing neurons were quantified relative to those for control pLSyn vector-containing neurons. (C) Quantitative RT-PCR for APP and RanBP9 mRNA, using hippocampal cell total RNA as the template. Expression levels in miR-101 sponge-transduced neurons relative to control pLSyn vector-transduced cells are shown. (D) Conditioned culture medium samples from lentivirus-transduced hippocampal neurons were collected from cells cultured in 35-mm dishes, normalized according to the protein content of the corresponding cell extracts, and analyzed by Western blotting with the sAPPβ antibody, which recognizes the secreted form of APP. Cultures expressing the pLSyn-miR-101 sponge exhibited sAPPβ levels that were 1.3-fold higher than the control levels found in pLSyn-transduced cells. Results in (B–D) are presented as the means ± the SE of three independent experiments ( * p < 0.05, Student's t -test).

Article Snippet: The SC-RANBP9 3′UTR was purchased from Origene (SC210772).

Techniques: Inhibition, Western Blot, Plasmid Preparation, Infection, Quantitative RT-PCR, Expressing, Cell Culture

Journal: Frontiers in Cellular Neuroscience

Article Title: A lentiviral sponge for miR-101 regulates RanBP9 expression and amyloid precursor protein metabolism in hippocampal neurons

doi: 10.3389/fncel.2014.00037

Figure Lengend Snippet: Silencing of RanBP9 reverses oversecretion of sAPPβ into the conditioned medium of pLSyn-miR-101-containing hippocampal neurons . Hippocampal neurons at 7 days in vitro were infected with the control pLSyn vector or the pLSyn-miR-101 sponge vector. Seventy-two hours later, the cells were transduced with a lentiviral vector containing RanBP9 siRNA under the control of the U6 promoter, or with a control lentivirus expressing a scrambled siRNA. After 14 days, the neurons were collected for Western blot analysis. (A) Representative immunoblots of protein extracts from lentiviral vector-transduced neurons reacted with an antibody recognizing RanBP9, an antibody recognizing full-length APP (4G8), or an antibody recognizing GAPDH are shown. Alternatively, conditioned culture medium samples were collected from lentiviral vector-transduced hippocampal neurons cultured in 35-mm dishes, normalized according to the protein content of the corresponding cell extracts, and analyzed by Western blotting with the sAPPβ antibody. (B) Quantitative data are provided in the histogram. The data are expressed as means ± the SE of three independent experiments ( * p < 0.05, Student's t -test).

Article Snippet: The SC-RANBP9 3′UTR was purchased from Origene (SC210772).

Techniques: In Vitro, Infection, Plasmid Preparation, Transduction, Expressing, Western Blot, Cell Culture

Journal: Frontiers in Cellular Neuroscience

Article Title: A lentiviral sponge for miR-101 regulates RanBP9 expression and amyloid precursor protein metabolism in hippocampal neurons

doi: 10.3389/fncel.2014.00037

Figure Lengend Snippet: Full-length APP and RanBP9 are targets of miR-101 in mouse hippocampal neurons in vivo . Western blotting was performed of hippocampal tissues injected and non-injected with lentiviral vectors. Effective expression of the lentiviral vector was revealed by the presence of EGFP (data not shown). Expression of the miR-101 lentiviral sponge resulted in an increase in the protein expression levels of the miR-101 targets, APP and RanBP9, with respect to non-injected tissue. APP and RanBP9 band intensities were quantified by densitometry, normalized to the GAPDH signal, and expressed as arbitrary OD units. The relative ratio of APP (pLSyn n = 3; pLSyn-miR-101 sponge n = 4) and RanBP9 (pLSyn n = 3; pLSyn-miR-101 sponge n = 4) expression in injected vs. non-injected tissue is shown. Results are presented as the means ± the SE ( * p < 0.05; ** p < 0.01, Student's t -test).

Article Snippet: The SC-RANBP9 3′UTR was purchased from Origene (SC210772).

Techniques: In Vivo, Western Blot, Injection, Expressing, Plasmid Preparation