human pre mir 497 195  (Addgene inc)

Journal: Oncotarget

Article Title: miR-195 in human primary mesenchymal stromal/stem cells regulates proliferation, osteogenesis and paracrine effect on angiogenesis

doi:

Figure Lengend Snippet: A. miR-195 and miR-497 levels after electroporation of human MSC with miR-195 and miR-497 were determined by quantitative real time PCR. Small nuclear RNA U6 was used as reference gene. Values shown represent 2 independent experiments and are relative to scrambled negative control (SCR). B. ALP staining 7 days after electroporation of human MSC with scrambled negative control (SCR), miR-195 or miR-497 (5X; microscope scale: 50 μm; photography scale: 2 mm). C. ALP and RUNX2 expression levels in human MSC electroporated with either SCR, miR-195, miR-497. D. ALP expression levels 48h after MSC transfection with SCR negative control or anti-miR-195; ALP staining quantification in MSC transfected with SCR negative control or anti-miR-195 after 5 days of incubation with osteogenic supplements. Values shown represent 2 independent experiments and are relative to SCR (mean±SD; * P < 0.05, Student t test).

Article Snippet: MC3T3 cells (3×10 6 ) plated in a 10-cm culture dish were transfected with 50 nM Pre-miR miRNA Precursors miR-143, Pre-miR miRNA Precursors miR-195, Pre-miR miRNA Precursors miR-497 or Pre-miR miRNA Precursor Negative Control (Scrambled - SCR) (Life Technologies) using Lipofectamine 2000 transfection reagent (Invitrogen, Life Technologies).

Techniques: Electroporation, Real-time Polymerase Chain Reaction, Negative Control, Staining, Microscopy, Expressing, Transfection, Incubation

Journal: Oncotarget

Article Title: miR-195 in human primary mesenchymal stromal/stem cells regulates proliferation, osteogenesis and paracrine effect on angiogenesis

doi:

Figure Lengend Snippet: A. A representative experiment on cell viability of miR-195 and miR-497 in human MSC. Fluorescence of resorufin was measured every 24 hours for 4 days post-electroporation with SCR, miR-195 or miR-497. Values represent the mean±SD of 5 replicates (*** P < 0 .001, Student t test). Two independent experiments were performed. B. Confocal imaging of MSC cells 48 hours after electroporation of SCR, miR-195 or miR-497. Cells were stained for the proliferation marker Ki-67 protein (red); nuclear DNA was labeled with DAPI (blue). Differences in the percentage of cells in proliferation (Ki-67+/DAPI+) and the average number of nuclei (DAPI+) were determined in 2 independent experiments by analyzing in at least 5 different images per condition with a minimum of 100 DAPI+ nuclei each (* P < 0.05, one-way ANOVA). Laser Scanning Spectral Confocal Microscope Leica TCS SP2 was used. A representative merged fluorescence image (20X objective, zoom factor of 5) per condition is shown (DAPI+ - blue; Ki-67+ - red). Scale: 40 μm. C. Differences in the percentage of proliferative MSC (Ki-67+/DAPI+) 96h after transfection of SCR negative control or anti-miR-195 (* P < 0.05, one-way ANOVA).

Article Snippet: MC3T3 cells (3×10 6 ) plated in a 10-cm culture dish were transfected with 50 nM Pre-miR miRNA Precursors miR-143, Pre-miR miRNA Precursors miR-195, Pre-miR miRNA Precursors miR-497 or Pre-miR miRNA Precursor Negative Control (Scrambled - SCR) (Life Technologies) using Lipofectamine 2000 transfection reagent (Invitrogen, Life Technologies).

Techniques: Fluorescence, Electroporation, Imaging, Staining, Marker, Labeling, Microscopy, Transfection, Negative Control

Journal: Oncotarget

Article Title: miR-195 in human primary mesenchymal stromal/stem cells regulates proliferation, osteogenesis and paracrine effect on angiogenesis

doi:

Figure Lengend Snippet: A. Number of vessels in the CAM after 72 hours incubation with SCR-electroporated MSC condition media or miR-195-electroporated MSC condition media (mean±SD, ** P < 0.01, Student t test). Graphic includes results from 3 independent replicates, in a total of 42 analyzed eggs. B. Three representative images are shown per condition (20X; scale: 1mm). C. Number of vessels in the CAM after 72 hours incubation with SCR-electroporated MSC conditioned media or miR-195-electroporataed MSC conditioned media with or without human recombinant VEGF supplementation (mean±SD, * P < 0.05, one way ANOVA), in a total of 34 analyzed eggs.

Article Snippet: MC3T3 cells (3×10 6 ) plated in a 10-cm culture dish were transfected with 50 nM Pre-miR miRNA Precursors miR-143, Pre-miR miRNA Precursors miR-195, Pre-miR miRNA Precursors miR-497 or Pre-miR miRNA Precursor Negative Control (Scrambled - SCR) (Life Technologies) using Lipofectamine 2000 transfection reagent (Invitrogen, Life Technologies).

Techniques: Incubation, Recombinant

Journal: Oncotarget

Article Title: miR-195 in human primary mesenchymal stromal/stem cells regulates proliferation, osteogenesis and paracrine effect on angiogenesis

doi:

Figure Lengend Snippet: A. VEGF expression levels are decreased in miR-195-overexpressing MSC compared with scrambled when analyzed by quantitative real-time PCR (** P < 0.01, Student t test). VEGF expression levels are increased in anti-miR-195 MSC compared with scrambled negative control when analyzed by quantitative real-time PCR (* P < 0.05, Student t test). B. Levels of secreted VEGF protein (pg/ml) were measured by enzyme-linked immunosorbent assay and are reduced in miR-195-MSC conditioned media versus SCR-MSC conditioned media (* P < 0.05, Student t test), while increased in anti-miR-195-MSC conditioned media versus SCR negative control-MSC conditioned media (* P < 0.05, Student t test). C. Luciferase levels were reduced when PGL3-VEGF (VEGF) but not PGL3-VEGF-mutated (VEGF_Mut) was cotransfected with miR-195 compared with scrambled (SCR), in both U-2 OS and HeLa cells. Values represent mean±SD of 3 replicates (* P < 0.05, Student t test). D. Luciferase levels were not altered after cotranfection of PGL3-SMAD5 (SMAD5) with miR-195 compared with scrambled (SCR) in U-2OS cells. E. Luciferase levels were not altered after cotranfection of PGL3-HOXA10 (HOX10A) with miR-195 compared with scrambled (SCR) in U-2OS cells. F. Expression levels of SMAD5 were decreased 48hours after MSC miR-195-electroporation, while increased 48hours after MSC anti-miR-195-transfection compared with control. Values represent mean±SD of 2 replicates (* P < 0.05, Student t test). G. Expression levels of HOXA10 were decreased 48hours after MSC miR-195-electroporation, while increased 48hours after MSC anti-miR-195-transfection compared with control. Values represent mean±SD of 2 replicates (* P < 0.05, Student t test).

Article Snippet: MC3T3 cells (3×10 6 ) plated in a 10-cm culture dish were transfected with 50 nM Pre-miR miRNA Precursors miR-143, Pre-miR miRNA Precursors miR-195, Pre-miR miRNA Precursors miR-497 or Pre-miR miRNA Precursor Negative Control (Scrambled - SCR) (Life Technologies) using Lipofectamine 2000 transfection reagent (Invitrogen, Life Technologies).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Negative Control, Enzyme-linked Immunosorbent Assay, Luciferase, Electroporation, Transfection