Journal: bioRxiv

Article Title: Transcriptomic Profiling of Skeletal Muscle Adaptations to Exercise and Inactivity

doi: 10.1101/813048

Figure Lengend Snippet: A) Genes significantly modified by acute aerobic and resistance exercise and inactivity were overlapped in a Venn Diagram. B) DNAJA4 , KLHL40 , NR4A3 and VGLL2 were validated in an independent cohort of pre- and post-acute aerobic exercise. Individual paired t -tests vs pre, n =8, **p<0.01. C) DNAJA4 , NR4A3 , KLHL40 and VGLL2 gene expression following electrical pulse stimulation in primary human myotubes. Individual paired t -tests vs basal, n =8, **p<0.01.

Article Snippet: Antibodies were directed to NR4A3/NOR1 (Dilution 1:1000, NBP2-46246, Novus Biologicals) and mitochondrial complexes (Dilution 1:1000, Total OXPHOS Human WB Antibody Cocktail, Abcam Cat# ab110411, RRID:AB_2756818).

Techniques: Modification, Expressing

Journal: bioRxiv

Article Title: Transcriptomic Profiling of Skeletal Muscle Adaptations to Exercise and Inactivity

doi: 10.1101/813048

Figure Lengend Snippet: A) NR4A3 responds in a time- and intensity-dependent manner to electrical pulse stimulation. n =3, 2-way ANOVA (time, intensity), *overall effect p<0.05. B) Silencing efficiency using siRNA against NR4A3. C) Electrical pulse stimulation-induced glucose uptake is abolished after NR4A3 silencing. 2-way ANOVA (si NR4A3 , EPS), n =6, *p<0.05. D) Silencing NR4A3 using siRNA modifies the mRNA levels of exercise- and inactivity-responsive genes. n =4, individual paired t -test vs scramble, ***p<0.001. E) Silencing of NR4A3 correlates with inactivity observed in MetaMEx. F) Reduction of NR4A3 level impairs basal and maximal oxygen consumption measured by Seahorse XF analysis. Individual paired t -tests vs scramble, n =5, *p<0.05, **p<0.01. G) Silencing of NR4A3 leads to a drift of muscle cells towards a more quiescent phenotype. H) Silencing of NR4A3 decreases the abundance of mitochondrial complexes. Representative blot and quantification, n =4, individual paired t -tests vs scramble, **p<0.01. I) The increase in glycolysis (ECAR) induced by beta-adrenergic stimulation (20 µM salbutamol for 3h) is impaired in the absence of NR4A3 . SeaHorse XF experiment, 2-way ANOVA (si NR4A3 , Salbutamol), n =7, **p<0.01. AA: Acute Aerobic, AR: Acute Resistance, IN: Inactivity, TA: Training Aerobic, TR: Training Resistance.

Article Snippet: Antibodies were directed to NR4A3/NOR1 (Dilution 1:1000, NBP2-46246, Novus Biologicals) and mitochondrial complexes (Dilution 1:1000, Total OXPHOS Human WB Antibody Cocktail, Abcam Cat# ab110411, RRID:AB_2756818).

Techniques:

Journal: bioRxiv

Article Title: Transcriptomic Profiling of Skeletal Muscle Adaptations to Exercise and Inactivity

doi: 10.1101/813048

Figure Lengend Snippet: NR4A3 was silenced using siRNA. Cells were exposed to 20 µM of salbutamol for 3h before analyzing oxygen consumption and extracellular acidification rate using the seahorse technology.

Article Snippet: Antibodies were directed to NR4A3/NOR1 (Dilution 1:1000, NBP2-46246, Novus Biologicals) and mitochondrial complexes (Dilution 1:1000, Total OXPHOS Human WB Antibody Cocktail, Abcam Cat# ab110411, RRID:AB_2756818).

Techniques:

Journal: Open Biology

Article Title: TNF-α-induced NF-κB activation upregulates microRNA-150-3p and inhibits osteogenesis of mesenchymal stem cells by targeting β-catenin

doi: 10.1098/rsob.150258

Figure Lengend Snippet: TNF-α treatment upregulated miR-150-3p and downregulated β-catenin expressions in hBM-MSCs. ( a ) Expressions of miR-150-3p in hBM-MSCs treated with either PBS (as control) or TNF-α. ( b ) Expressions of several genes known to be regulated by TNF-α in hBM-MSCs after the same treatment as in ( a ). ( c ) Expressions of genes from ( b ) in hBM-MSCs transfected with either miR-NC (negative control) or miR-150-3p. Data were presented as mean ± s.d. from at least three independent experiments. ** p < 0.01, * p < 0.05 compared with respective PBS or miR-NC control.

Article Snippet: Human miR-150-3p mir Vana miRNA mimic and antisense oligos (MH12324) were purchased from Applied Biosystems (Carlsbad, CA, USA), and transfected into cells according to the manufacturer's instructions.

Techniques: Control, Transfection, Negative Control

Journal: Open Biology

Article Title: TNF-α-induced NF-κB activation upregulates microRNA-150-3p and inhibits osteogenesis of mesenchymal stem cells by targeting β-catenin

doi: 10.1098/rsob.150258

Figure Lengend Snippet: TNF-α treatment upregulated miR-150-3p, which directly targeted the 3′-UTR on β-catenin mRNA in hBM-MSCs. ( a ) Sequence of the putative miR-150-3p targeting site (capitalized nucleotides) on the 3′-UTR of β-catenin mRNA. ( b ) At the downstream of a luciferase reporter open reading frame (LUF), wild-type (-WT) or mutated (-Mut, grey nucleotides) versions of putative targeting sequence from the 3′-UTR of β-catenin mRNA were cloned. ( c ) Luciferase activities of LUF-WT and LUF-Mut constructs were measured in hBM-MSCs co-transfected with either miR-NC (negative control) or miR-150-3p. Data presented as mean ± s.d. from at least three independent experiments. ** p < 0.01, * p < 0.05 compared with respective PBS or miR-NC control. ( d ) Luciferase activities of LUF-WT and LUF-Mut constructs were measured in hBM-MSCs treated with either PBS (as control) or TNF-α. ( e ) β-catenin protein levels in hBM-MSCs transfected with either miR-NC or miR-150-3p were examined by Western blot. ( f ) hBM-MSCs were transfected with either miR-NC or miR-150-3p inhibitor (anti-miR), and treated with either PBS (−) or TNF-α (+), followed by Western blot analysis to examined β-catenin protein levels.

Article Snippet: Human miR-150-3p mir Vana miRNA mimic and antisense oligos (MH12324) were purchased from Applied Biosystems (Carlsbad, CA, USA), and transfected into cells according to the manufacturer's instructions.

Techniques: Sequencing, Luciferase, Clone Assay, Construct, Transfection, Negative Control, Control, Western Blot

Journal: Open Biology

Article Title: TNF-α-induced NF-κB activation upregulates microRNA-150-3p and inhibits osteogenesis of mesenchymal stem cells by targeting β-catenin

doi: 10.1098/rsob.150258

Figure Lengend Snippet: NF-κB-induced miR-150-3p expression by directly binding to its promoter region. ( a ) Promoter region of human miR-150 gene contains three putative binding sites for NF-κB. At the upstream of a luciferase reporter open reading frame (LUF), segments of putative NF-κB binding sites from the miR-150 promoter were cloned. ( b ) Binding of NF-κB to the promoter of miR-150 in hBM-MSCs was examined by ChIP assay using IgG (as control) or anti-NF-κB antibody. ( c ) Luciferase activities of ABC-LUF, BC-LUF and C-LUF constructs were measured in hBM-MSCs treated with either PBS (as control) or NF-κB. Data presented as mean ± s.d. from at least three independent experiments. ** p < 0.01 compared with respective IgG or PBS control.

Article Snippet: Human miR-150-3p mir Vana miRNA mimic and antisense oligos (MH12324) were purchased from Applied Biosystems (Carlsbad, CA, USA), and transfected into cells according to the manufacturer's instructions.

Techniques: Expressing, Binding Assay, Luciferase, Clone Assay, Control, Construct

Journal: Open Biology

Article Title: TNF-α-induced NF-κB activation upregulates microRNA-150-3p and inhibits osteogenesis of mesenchymal stem cells by targeting β-catenin

doi: 10.1098/rsob.150258

Figure Lengend Snippet: TNF-α treatment and miR-150-3p transfection both inhibited osteogenic differentiation of hBM-MSCs. ( a ) Calcium content, alizarin red (ALR) and alkaline phosphatase (ALP) staining assays were measured in hBM-MSCs treated with either PBS (as control) or TNF-α. ( b,c ) Representative images of ALR and ALP stainings ( b ), and expressions of Runx2 and Osx ( c ) after the same treatment as in ( a ). ( d ) Calcium content, alizarin red (ALR) and alkaline phosphatase (ALP) staining assays were measured in hBM-MSCs transfected with either miR-NC (negative control) or miR-150-3p. ( e,f ) Representative images of ALR and ALP stainings ( e ), and expressions of Runx2 and Osx ( f ) after the same treatment as in ( d ). Data presented as mean ± s.d. from at least three independent experiments. ** p < 0.01, * p < 0.05 compared with respective PBS or miR-NC control.

Article Snippet: Human miR-150-3p mir Vana miRNA mimic and antisense oligos (MH12324) were purchased from Applied Biosystems (Carlsbad, CA, USA), and transfected into cells according to the manufacturer's instructions.

Techniques: Transfection, Staining, Control, Negative Control

Journal: Open Biology

Article Title: TNF-α-induced NF-κB activation upregulates microRNA-150-3p and inhibits osteogenesis of mesenchymal stem cells by targeting β-catenin

doi: 10.1098/rsob.150258

Figure Lengend Snippet: IKK activation was essential for TNF-α induced upregulation of miR-150-3p in hBM-MSCs. ( a ) Expressions of miR-150-3p in hBM-MSCs treated with p38 MAPK inhibitor BIX02188, JNK inhibitor SP600125 and IKK inhibitor TPCA-1, respectively, in the presence of TNF-α. ( b ) NF-κB protein and ( c ) miR-150-3p levels in hBM-MSCs transfected with siRNAs against NF-κB were examined. ( d ) β-catenin protein levels in hBM-MSCs treated with small inhibitors (left panels) or siRNAs against NF-κB (right panels) in the presence of TNF-α were examined with Western blot. ( e ) Levels of phosphorylated NF-κB at Ser536 (p-NF-κB) in hBM-MSCs treated with either PBS (as control) or TNF-α. Data presented as mean ± s.d. from at least three independent experiments. ** p < 0.01 compared with respective Mock or siRNA-NC control.

Article Snippet: Human miR-150-3p mir Vana miRNA mimic and antisense oligos (MH12324) were purchased from Applied Biosystems (Carlsbad, CA, USA), and transfected into cells according to the manufacturer's instructions.

Techniques: Activation Assay, Transfection, Western Blot, Control

Journal: Open Biology

Article Title: TNF-α-induced NF-κB activation upregulates microRNA-150-3p and inhibits osteogenesis of mesenchymal stem cells by targeting β-catenin

doi: 10.1098/rsob.150258

Figure Lengend Snippet: MiR-150-3p was required for TNF-α-induced inhibition of hBM-MSC osteogenic differentiation. ( a ) hBM-MSCs were transfected with either miR-NC (as control) or miR-150-3p inhibitor (anti-miR), then treated with either PBS (as control) or TNF-α, followed by measurements of calcium content, alizarin red (ALR) and alkaline phosphatase (ALP) stainings. ( b,c ) Representative images of ALR and ALP stainings ( b ), and expressions of Runx2 and Osx ( c ) after the same treatment as in ( a ). For the easy of comparison, results from figure 5 (PBS and TNF-α treatments) were also plotted. Data presented as mean ± s.d. from at least three independent experiments. ** p < 0.01, * p < 0.05 compared with respective PBS control. ## p < 0.01, # p < 0.05 compared with respective miR-NC control.

Article Snippet: Human miR-150-3p mir Vana miRNA mimic and antisense oligos (MH12324) were purchased from Applied Biosystems (Carlsbad, CA, USA), and transfected into cells according to the manufacturer's instructions.

Techniques: Inhibition, Transfection, Control, Comparison