expression vectors coding for myod and runx1  (Addgene inc)

Journal: International Journal of Molecular Sciences

Article Title: Phosphodiesterase 5a Signalling in Skeletal Muscle Pathophysiology

doi: 10.3390/ijms24010703

Figure Lengend Snippet: Modulation of Pde5a1 by MYOD and RUNX1. ( a ) Scheme of Pde5A promoter constructs transfected in cells. C2C12 cells were transiently co-transfected with a plasmid expressing MyoD ( b ) or Runx1 ( c ) and the different Pde5a1 promoter constructs. Results are the means of at least three different experiments performed in triplicate and expressed as the fold change activity relative to that observed in cells not overexpressing MyoD or Runx1. ( d ) C2C12 cells were transiently transfected with a plasmid expressing MyoD or Runx1 and the levels of Pde5a1 RNA were measured by RT-qPCR. Relative expression of Pde5a1 in cells overexpressing MYOD (dotted column) or RUNX1 (grey column) are expressed as the fold change compared to untreated cells (empty columns) All values represent the mean ± SD of n = 3 different RNA preparations. * Indicates statistical significance by the t -test ( p < 0.05).

Article Snippet: Expression vectors coding for MyoD and Runx1 were purchased from Addgene (Watertown, MA, USA).

Techniques: Construct, Transfection, Plasmid Preparation, Expressing, Activity Assay, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Phosphodiesterase 5a Signalling in Skeletal Muscle Pathophysiology

doi: 10.3390/ijms24010703

Figure Lengend Snippet: MyoD and Runx1 mRNA levels in skeletal muscle under pathophysiological conditions. The mRNA levels of MyoD and Runx1 were measured by RT-qPCR. mRNA levels in the samples (dotted or grey columns) are expressed as the fold change compared with the samples from muscle from wild-type mice (empty columns). All values represent the mean ± SD of n = 3 different RNA preparations. * Indicates statistical significance of pathological samples compared with control samples ( t -test: p < 0.05).

Article Snippet: Expression vectors coding for MyoD and Runx1 were purchased from Addgene (Watertown, MA, USA).

Techniques: Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: Phosphodiesterase 5a Signalling in Skeletal Muscle Pathophysiology

doi: 10.3390/ijms24010703

Figure Lengend Snippet: Primers used in RNA analysis and promoters cloning.

Article Snippet: Expression vectors coding for MyoD and Runx1 were purchased from Addgene (Watertown, MA, USA).

Techniques: Cloning

Journal: Scientific reports

Article Title: Mohawk impedes angiofibrosis by preventing the differentiation of tendon stem/progenitor cells into myofibroblasts.

doi: 10.1038/s41598-022-24195-5

Figure Lengend Snippet: Figure 3. MyoD induces TSPCs to myofibroblasts reprogramming in vitro. (A) and (B) Acta2 and Col3a1 qRT-PCR quantification comparing 3 conditions: “control TSPCs”, “MyoD-expressing TSPCs (14 days after transfection)” and “TSPCs + MyoD + Xav939 treatment”. N = 3. (C) Representative morphological change 14 days after MyoD expression under inverted (scale bar, 50 μm) or scanning electron microscope (scale bar, 25 μm). (D) PrestoBlue cell viability assay of MyoD-expressing or control TSPCs. ***p < 0.001. (E) Cellular length comparison and (F) width (Control vs. MyoD-expressing TSPCs 14 days after transfection) using image J software, 20 microscopic fields, **p < 0.01. (G) FACS gating strategy for control TSPCs and (H) myofibroblasts. (I) FACS based quantification of ACTA2 + cells. *p < 0.05. N = 3. (J) Representative immunofluorescence images of MyoD-expressing TSPCs vs. control TSPCs stained for VEGF (green) and α-SMA (red). N = 3. Scale bar, 20 μm.

Article Snippet: Neonatal TSPCs were transfected at 60–70% confluence with MyoD expressing vectors (CMV-MyoD, a gift from Andrew Lassar, Addgene plasmid # 8398) or with empty vectors (controls, CMV-MyoD restricted using EcoRI and ligated) using lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s recommendations.

Techniques: In Vitro, Quantitative RT-PCR, Control, Expressing, Transfection, Microscopy, Viability Assay, Comparison, Software, Immunofluorescence, Staining

Journal: Scientific reports

Article Title: Mohawk impedes angiofibrosis by preventing the differentiation of tendon stem/progenitor cells into myofibroblasts.

doi: 10.1038/s41598-022-24195-5

Figure Lengend Snippet: Figure 5. In-vivo Wnt signaling modulation during adult tendon healing. (A) qRT-PCR Acta2 and Col3a1 quantification comparing 3 conditions: (1) control TSPCs (2) MyoD-expressing TSPCs (3) TSPCs + MyoD + Xav939 treatment. N = 3. (B) Representative H&E and Masson’s trichrome staining images of control and Xav939-treated tendons d28 after injury. N = 6 tendons/group. Scale bar, 40 μm, and (C) the corresponding Collagen deposition quantification. (D) Width quantification of control and Xav939-treated Achilles tendons (20 zones along 5 tendons/group). (E) Tendon thickness of control and Xav939-treated tendons (20 zones from 5 tendons/group). (F–I) Maximum force, yield elongation, stiffness and maximum tensile stress properties in the control and Xav939-treated groups d52 after injury. N = 6. Representative immunofluorescence staining images of α-SMA and VEGF.N = 5 tendons/group. Scale bar, 40 μm.

Article Snippet: Neonatal TSPCs were transfected at 60–70% confluence with MyoD expressing vectors (CMV-MyoD, a gift from Andrew Lassar, Addgene plasmid # 8398) or with empty vectors (controls, CMV-MyoD restricted using EcoRI and ligated) using lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s recommendations.

Techniques: In Vivo, Quantitative RT-PCR, Control, Expressing, Staining, Immunofluorescence

Journal: Scientific Reports

Article Title: Mohawk impedes angiofibrosis by preventing the differentiation of tendon stem/progenitor cells into myofibroblasts

doi: 10.1038/s41598-022-24195-5

Figure Lengend Snippet: MyoD induces TSPCs to myofibroblasts reprogramming in vitro. ( A ) and (B ) Acta2 and Col3a1 qRT-PCR quantification comparing 3 conditions: “control TSPCs”, “MyoD-expressing TSPCs (14 days after transfection)” and “TSPCs + MyoD + Xav939 treatment”. N = 3. ( C ) Representative morphological change 14 days after MyoD expression under inverted (scale bar, 50 μm) or scanning electron microscope (scale bar, 25 μm). ( D ) PrestoBlue cell viability assay of MyoD -expressing or control TSPCs. ***p < 0.001. ( E ) Cellular length comparison and ( F ) width (Control vs. MyoD-expressing TSPCs 14 days after transfection) using image J software, 20 microscopic fields, **p < 0.01. ( G ) FACS gating strategy for control TSPCs and ( H ) myofibroblasts. ( I ) FACS based quantification of ACTA2 + cells. *p < 0.05. N = 3. ( J ) Representative immunofluorescence images of MyoD-expressing TSPCs vs . control TSPCs stained for VEGF (green) and α-SMA (red). N = 3. Scale bar, 20 μm.

Article Snippet: Neonatal TSPCs were transfected at 60–70% confluence with MyoD expressing vectors (CMV- MyoD , a gift from Andrew Lassar, Addgene plasmid # 8398) or with empty vectors (controls, CMV- MyoD restricted using EcoRI and ligated) using lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s recommendations.

Techniques: In Vitro, Quantitative RT-PCR, Control, Expressing, Transfection, Microscopy, Viability Assay, Comparison, Software, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: Mohawk impedes angiofibrosis by preventing the differentiation of tendon stem/progenitor cells into myofibroblasts

doi: 10.1038/s41598-022-24195-5

Figure Lengend Snippet: In-vivo Wnt signaling modulation during adult tendon healing. ( A ) qRT-PCR Acta2 and Col3a1 quantification comparing 3 conditions: (1) control TSPCs (2) MyoD-expressing TSPCs (3) TSPCs + MyoD + Xav939 treatment. N = 3. ( B ) Representative H&E and Masson’s trichrome staining images of control and Xav939-treated tendons d28 after injury. N = 6 tendons/group. Scale bar, 40 μm, and ( C ) the corresponding Collagen deposition quantification . ( D ) Width quantification of control and Xav939-treated Achilles tendons (20 zones along 5 tendons/group). ( E ) Tendon thickness of control and Xav939-treated tendons (20 zones from 5 tendons/group). ( F–I ) Maximum force, yield elongation, stiffness and maximum tensile stress properties in the control and Xav939-treated groups d52 after injury. N = 6. Representative immunofluorescence staining images of α-SMA and VEGF.N = 5 tendons/group. Scale bar, 40 μm.

Article Snippet: Neonatal TSPCs were transfected at 60–70% confluence with MyoD expressing vectors (CMV- MyoD , a gift from Andrew Lassar, Addgene plasmid # 8398) or with empty vectors (controls, CMV- MyoD restricted using EcoRI and ligated) using lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s recommendations.

Techniques: In Vivo, Quantitative RT-PCR, Control, Expressing, Staining, Immunofluorescence

Journal: Neuromuscular Disorders

Article Title: Multiple exon skipping strategies to by-pass dystrophin mutations

doi: 10.1016/j.nmd.2011.10.007

Figure Lengend Snippet: Alternative strategies for intra-exonic mutations. (A) Locations of AO annealing sites in human dystrophin exons 50, 51 and 52, the location of the 7348DupG mutation and the two options to correct a frame-shifting mutation in exon 51 are indicated: (B) skipping exons 50&51 and (C) skipping exons 51&52. Nested RT-PCR across exons 48–55 showing skipping of exons (D) 50&51 and (E) 51&52 in MyoD transformed patient fibroblasts. Full-length (FL) transcript is 1088 bp, Δ50&51 transcript is 745 bp and Δ51&52 transcript is 736 bp. (F) Locations of AO annealing sites in human dystrophin exons 19, 20 and 21, and the location of the 2 and 8 base deletions. Two options to correct these frame-shifting mutations in exon 20: (G) skipping exons 19&20 and (H) skipping exons 20&21. Nested RT-PCR across exons 17–25 shows skipping of exons 19&20 in (I) 2 base deletion cells and (K) 8 base deletion cells, and skipping of exons 20&21 in (J) in 2 base deletion cells and (L) 8 base deletion cells. Marker is a 100 bp ladder. Full-length (FL) transcript is 1255 bp, Δ19&20 transcript is 925 bp, Δ20&21 transcript is 832 bp and Δ21&22 transcript is 928 bp.

Article Snippet: Fibroblast cultures were first transformed to a myogenic lineage with a MyoD expressing adenoviral vector (The Native Antigen Company, Oxford, UK) , at an MOI of 200 and allowed to differentiate for 2–3 days, once multinucleated myotubes were observed, prior to transfection.

Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Marker

Journal: Neuromuscular Disorders

Article Title: Multiple exon skipping strategies to by-pass dystrophin mutations

doi: 10.1016/j.nmd.2011.10.007

Figure Lengend Snippet: Alternative strategies for exon 21 and 22 splice site mutations. (A) A single A>G mutation at the exon 22 acceptor splice site induces retention of a single G nucleotide from intron 21. The reading frame is restored by (B) excision of exons 21&22 or (C) excision of exon 21 only. Nested RT-PCR across exons 17–25 showing (D) skipping of exons 21&22 or (E) exon 21 only in MyoD transformed patient fibroblasts with total AO concentrations of 600–25 nM. (F) A single C>G mutation at the exon 21 acceptor splice site allows retention of 2 nucleotides from intron 20. The reading frame is restored by exclusion of (G) skipping of exons 20&21, or (H) exon 20 alone. Nested RT-PCR across exons 17–25 showing skipping of (I) exons 20&21 and (J) exon 20 only, at total AO concentrations of 600–25 nM. Marker is a 100 bp ladder. Full-length (FL) transcript is 1255 bp, Δ20 transcript is 1013 bp, Δ21 transcript is 1074 bp and Δ20&21 is 832 bp.

Article Snippet: Fibroblast cultures were first transformed to a myogenic lineage with a MyoD expressing adenoviral vector (The Native Antigen Company, Oxford, UK) , at an MOI of 200 and allowed to differentiate for 2–3 days, once multinucleated myotubes were observed, prior to transfection.

Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Marker