Journal: International Journal of Molecular Sciences

Article Title: Tenogenic Contribution to Skeletal Muscle Regeneration: The Secretome of Scleraxis Overexpressing Mesenchymal Stem Cells Enhances Myogenic Differentiation In Vitro

doi: 10.3390/ijms21061965

Figure Lengend Snippet: Immunohistochemistry against myosin heavy chain 1E (MYH1E) (turquoise) after 5 days of myogenic differentiation showed newly fused myotubes in all culture conditions ( A–A’’ , arrowheads). However, myoblasts that were exposed to conditioned medium (CM) derived from SCP1 GFP/SCX cells showed an increased fusion, when compared to C2C12 in NM or CM of SCP1 GFP cells (A-A’’). Quantification of the MYH1E positive area ( B ) and the fusion index ( C ) validated the microscopic appearance. Scale bars: 500 µm (overview), 200 µm (insert). Bar plots represent the mean and SD. ** equals p ≤ 0.01, *** equals p ≤ 0.001. Five randomly selected pictures of three independent experiments were analyzed.

Article Snippet: Five days after the first CM exposure, newly formed myotubes were visualized by immunohistochemistry against myosin heavy chain 1E (MYH1E, clone MF20, obtained from the Developmental Studies Hybridoma Bank (DSHB) developed under the auspices of the National Institute of Child Health and Human Development (NICHD) and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242).

Techniques: Immunohistochemistry, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Tenogenic Contribution to Skeletal Muscle Regeneration: The Secretome of Scleraxis Overexpressing Mesenchymal Stem Cells Enhances Myogenic Differentiation In Vitro

doi: 10.3390/ijms21061965

Figure Lengend Snippet: Immunohistochemistry against MYH1E (turquoise) after 7 days of myogenic differentiation in co-culture with SCP1 GFP or SCP1 GFP/SCX cell pellets revealed that most of the newly formed myofibers were orientated towards the middle of the cell pellets ( A ). However, the quantification of the orientation did not show any significant difference between both groups ( B ). Vertical lines in violin plots represent the median (red) and quartiles (black). Red dashed lines indicate a perfect orientation towards the center of the pellet. n.s.: not significant. Scale bar: 500 µm.

Article Snippet: Five days after the first CM exposure, newly formed myotubes were visualized by immunohistochemistry against myosin heavy chain 1E (MYH1E, clone MF20, obtained from the Developmental Studies Hybridoma Bank (DSHB) developed under the auspices of the National Institute of Child Health and Human Development (NICHD) and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242).

Techniques: Immunohistochemistry, Co-Culture Assay

Journal: Blood

Article Title: The gene encoding the transcriptional regulator Yin Yang 1 (YY1) is a myeloid transforming gene interfering with neutrophilic differentiation.

doi: 10.1182/blood-2002-04-1207

Figure Lengend Snippet: Figure 3. Western blot analysis of YY1 expression in 32D-WT1 cells. G-CSF responsive 32D-WT1 cells were cultured in IL-3–containing medium and then switched to G-CSF. Samples were taken daily and processed as described in “Materials and methods.” The blot was hybridized with anti-YY1 or anti-Sp1, stripped, and rehybridized with anti-Actin to check for equal loading of cell lysates.

Article Snippet: Lysates of 32D cells were prepared and subjected to Western blotting as described previously.32 Antibodies used to visualize YY1 were goat anti-YY1 (Santa Cruz Biotechnology, CA) or rabbit anti-HA (Y-11, sc-805) for HA-tagged YY1.

Techniques: Western Blot, Expressing, Cell Culture

Journal: Blood

Article Title: The gene encoding the transcriptional regulator Yin Yang 1 (YY1) is a myeloid transforming gene interfering with neutrophilic differentiation.

doi: 10.1182/blood-2002-04-1207

Figure Lengend Snippet: Figure 1. Identification of common virus integrations in the YY1 locus. (A) Inverse PCR. Genomic DNA from leukemia cells was digested with HhaI. After ligation PCR was performed with primers L1 and L2 followed by a nested PCR with primers L1N and L2N to amplify LTR-flanking fragments from circularized DNA. (B) Nested PCR with LTR and YY1 primers to detect position and orientation of Graffi-1.4 MuLV integration in the YY1 promoter region. The lowercase letters indicate the position at the promoter in base pairs (bp). The first PCR was performed with the primer sets L1, Y1 (a), L1, Y2 (b), L2, Y1 (c), and L2, Y2 (d), followed by a nested PCR with primers L1N, Y1N (a) and L1N, Y2N (b), L2N, Y1N (c), and L2N, Y2N (d). Probes Y1P and Y2P were used to analyze the specificity of the PCR band by Southern blot. (C) Example of Southern blot analysis to determine virus integration and orientation in the 5 region of the YY1 gene. Results depicted are from DNA samples of leukemias 1, 2, 5, and 6. PCR products from the 4 different primer combinations (a, b, c, and d) were analyzed. In this example, the blot was hybridized with probe Y1P. The presence of the band in lane 1c indicates that tumor 1 has a virus integration in the reverse orientation. Tumor 5 has 2 YY1 virus integrations in the reverse orientation (lane 5c). Tumor 2 has an integration in the forward orientation (lane 2a). Tumor 6 harbors 2 integrations in both orientations (lanes 6a and 6c). All bands were sequenced to determine the exact location of virus integration. (D) Examples of virus integrations and orientations found in the YY1 promoter.

Article Snippet: Lysates of 32D cells were prepared and subjected to Western blotting as described previously.32 Antibodies used to visualize YY1 were goat anti-YY1 (Santa Cruz Biotechnology, CA) or rabbit anti-HA (Y-11, sc-805) for HA-tagged YY1.

Techniques: Virus, Inverse PCR, Ligation, Nested PCR, Southern Blot

Journal: Blood

Article Title: The gene encoding the transcriptional regulator Yin Yang 1 (YY1) is a myeloid transforming gene interfering with neutrophilic differentiation.

doi: 10.1182/blood-2002-04-1207

Figure Lengend Snippet: Figure 2. Effects of Graffi-1.4 LTR integration in the YY1 promoter region on gene transcription. Luciferase assays were performed in HEK 293 cells. One representative experiment of 3 is shown. Error bars represent the SD of the mean values of 3 independent experiments. Negative control: empty pGL3 vector. Reporter gene expression was calculated in arbitrary units, relative to -galactosidase expression.

Article Snippet: Lysates of 32D cells were prepared and subjected to Western blotting as described previously.32 Antibodies used to visualize YY1 were goat anti-YY1 (Santa Cruz Biotechnology, CA) or rabbit anti-HA (Y-11, sc-805) for HA-tagged YY1.

Techniques: Luciferase, Negative Control, Plasmid Preparation, Gene Expression, Expressing

Journal: Blood

Article Title: The gene encoding the transcriptional regulator Yin Yang 1 (YY1) is a myeloid transforming gene interfering with neutrophilic differentiation.

doi: 10.1182/blood-2002-04-1207

Figure Lengend Snippet: Figure 5. GM-CSF–induced colony formation by primary bone marrow cells after retroviral transduction of YY1. (A) RNA spot blot analysis of supernatants containing BABE/HA-YY1 or BABE vector control virus, showing that titers used for infection were comparable. The filter was hybridized with a BABE-specific cDNA probe. (B) GM-CFU assay of primary bone marrow progenitor cells following infection with BABE-HA-YY1 or BABE control virus. Bone marrow cells were plated in triplicate at densities of 10 to 50 103 cells per dish in 1 mL methylcellulose medium containing GM-CSF (20 U/mL) and puromycin (2.5 g/mL). Two independent experiments are shown.

Article Snippet: Lysates of 32D cells were prepared and subjected to Western blotting as described previously.32 Antibodies used to visualize YY1 were goat anti-YY1 (Santa Cruz Biotechnology, CA) or rabbit anti-HA (Y-11, sc-805) for HA-tagged YY1.

Techniques: Retroviral, Transduction, Plasmid Preparation, Control, Virus, Infection, Colony-forming Unit Assay

Journal: Blood

Article Title: The gene encoding the transcriptional regulator Yin Yang 1 (YY1) is a myeloid transforming gene interfering with neutrophilic differentiation.

doi: 10.1182/blood-2002-04-1207

Figure Lengend Snippet: Figure 4. Ectopic expression of HA-YY1 in 32D cells inhibits neutrophilic differentiation. (A) Western blot analysis with anti-YY1 in 32D-WT1 and with anti-HA antibodies in 32D-HA-YY1 cells after switching the cells from IL-3– to G-CSF– containing medium on t 0 days. The blot was reprobed with anti-actin antibodies for loading control. (B) Differential cell count (blasts, band form, and segmented nuclei) of 2 representative 32D-WT1 and 2 representative 32D-HA-YY1 clones. (C) Micro- graphs showing morphology of 32D-WT1 and 32D-HA-YY1 clones on day 5 (original magnification, 1000).

Article Snippet: Lysates of 32D cells were prepared and subjected to Western blotting as described previously.32 Antibodies used to visualize YY1 were goat anti-YY1 (Santa Cruz Biotechnology, CA) or rabbit anti-HA (Y-11, sc-805) for HA-tagged YY1.

Techniques: Expressing, Western Blot, Control, Cell Counting, Clone Assay

Journal: Blood

Article Title: The gene encoding the transcriptional regulator Yin Yang 1 (YY1) is a myeloid transforming gene interfering with neutrophilic differentiation.

doi: 10.1182/blood-2002-04-1207

Figure Lengend Snippet: Figure 6. Real-time quantitative PCR analysis of YY1 transcripts in 94 patients with AML. Data are relative to the mean expression in healthy bone marrow samples (n 6), with 95% confidence limits indicated by the horizontal lines. AML data represent the mean of 2 independent experiments. The 95% confidence interval was calculated as: Xmean (6 NBM samples) (1.96 SD).

Article Snippet: Lysates of 32D cells were prepared and subjected to Western blotting as described previously.32 Antibodies used to visualize YY1 were goat anti-YY1 (Santa Cruz Biotechnology, CA) or rabbit anti-HA (Y-11, sc-805) for HA-tagged YY1.

Techniques: Real-time Polymerase Chain Reaction, Expressing