Journal: bioRxiv

Article Title: Glycogen synthase kinase 3 alpha/beta deletion induces precocious growth plate remodeling and cell loss in mice

doi: 10.1101/2020.04.04.025700

Figure Lengend Snippet: Presence of Sox9 positive cells in Gsk3a/Gsk3b KO and control mice at P36, P43 and P58 was assessed by immunohistochemistry on frontal knee joint sections (A). Bone marrow cavity-like regions are highlighted in black dotted line. Localized expression and loss of GSK3alpha (B&C) and GSK3beta (D&E) in Gsk3a/Gsk3b KO mice was evaluated by IHC at P36 (B&D) and P43 (C&E). Images are representative of 4-7 mice per group for Sox9, and 3-4 mice per group were used for GSK3alpha and GSK3beta at both time points.

Article Snippet: Primary antibodies used for immunolabelling are as follows: goat polyclonal Sox9 (AF-3075, R&D Systems), rabbit monoclonal GSK3alpha (ab40870, Abcam), rabbit polyclonal GSK3beta (12456S, Cell Signaling Technologies), mouse monoclonal type II collagen (Col2; 10R-C135b, Fitzgerald), rabbit polyclonal osteocalcin (ab93876, Abcam), rabbit polyclonal beta-catenin (bs-1165R, Bioss Antibodies) and rabbit polyclonal GLI1 (PA5-72942, Invitrogen, Thermo Fisher).

Techniques: Control, Immunohistochemistry, Expressing

Journal: BMC developmental biology

Article Title: Sonic hedgehog promotes chondrogenesis of rabbit bone marrow stem cells in a rotary cell culture system.

doi: 10.1186/s12861-019-0198-4

Figure Lengend Snippet: Fig. 5 The effect of Shh on cartilage related markers during differentiation induction in the RCCS environment. a-h RT-PCR analysis of Sox9, ACAN, collagen II, collagen X, RUNX2, ALP, PPAR-γ and leptin on days 7,14 and 21 during differentiation induction. i, j Expression of Sox 9, collagen II, ACAN, collagen X, RUNX2, ALP and PPAR-γ in the RCCS dimensional environment on days 10 and 21 of chondrogenesis. The representative results were from three independent experiments. Significant differences between the control group are indicated by * p < 0.05, **p < 0.01; differences between Shh and TGF-β transfection groups are indicated by # p < 0.05 or ## p < 0.01

Article Snippet: Antibodies recognising Ptc (Aviva Systems Biology, San Diego, CA, USA; 1:500), Smo (Aviva Systems Biology; 1:1,600), Gli1 (Biorbyt, Cambridgeshire, UK; 1:1,000), Sox9 (OriGene, Rockville, MD, USA; 1:500), collagen II (Novus Biologicals, Littleton, CO, USA; 1:200), ACAN (Novus Biologicals; 1:100), collagen X (Abcam; 1:500), RUNX2 (Abcam; 1:500), ALP (Abcam; 1:500), PPAR-γ (Santa Cruz Biotechnology; 1:1000) and GAPDH (Novus Biologicals; 1:2000) were used as primary antibodies.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Transfection

Journal: International journal of molecular sciences

Article Title: Metabolic Reconfiguration Activates Stemness and Immunomodulation of PDLSCs.

doi: 10.3390/ijms23074038

Figure Lengend Snippet: Figure 3. Curcumin increased the capabilities of osteogenic and chondrogenic differentiation in PDLSCs. (A) Heat map of RNA-seq analysis data showing the PDLSC function-related genes that were differentially regulated by curcumin treatment. (B,C) qPCR assay showed the significantly elevated levels of osteoprogenitor markers (B) and chondroprogenitor markers (C) in curcumin treated PDLSCs. (D) Alizarin red staining showed that curcumin treated PDLSCs has increased capacity to form mineralized nodules under osteoinductive conditions. (E) Western blot analysis showed the expression levels of the osteogenic genes RUNX2 and ALP were greatly increased in curcumin treated PDLSCs under osteoinductive conditions. β-actin was used as a loading control. (F) Histological analysis showed chondrogenic differentiation of vehicle and curcumin treated PDLSCs. Scale bar, 25 µm. IF staining showed increased ACAN+ and SOX9+ cells after curcumin treatment in PDLSCs. Scale bar, 25 µm. * p < 0.005. Error bars represent the s.d. from the mean values.

Article Snippet: Anti-ACAN antibody was purchased from Abcam, Cambridge, UK, and anti-SOX9 antibody was purchased from Novus Biologicals, Littleton, CO, USA.

Techniques: RNA Sequencing, Staining, Western Blot, Expressing, Control

Journal: Scientific Reports

Article Title: Glial cell type-specific gene expression in the mouse cerebrum using the piggyBac system and in utero electroporation

doi: 10.1038/s41598-021-84210-z

Figure Lengend Snippet: In utero electroporation using the piggyBac system led to transgene expression in both neurons and glial cells in the mouse cerebrum. ( A ) Schematics of pPB-CAG-EGFP , pCAG-PBase and pCAG-mCherry plasmids. pPB-CAG-EGFP drives EGFP expression under the CAG promoter. pCAG-PBase has a piggyBac transposase, which recognizes the terminal region (TR) in piggyBac donor plasmids. ( B ) Experimental procedure. piggyBac plasmids ( pPB-CAG-EGFP and pCAG-PBase ) plus pCAG-mCherry were co-transfected into the mouse cerebrum using in utero electroporation at E15.5, and coronal sections were prepared at P30. ( C, D ) Coronal sections of the electroporated cerebrum. Higher magnification images corresponding to upper and lower layers of the cerebral cortex in ( C ) are shown in ( D ). In utero electroporation with the piggyBac system induced EGFP expression not only in mCherry-positive layer 2/3 neurons (arrows) but also in mCherry-negative cells (arrowheads). ( E–G ) Immunohistochemistry for NeuN ( E ), Sox9 and Sox10 ( F ), and in situ hybridization for Plp1 ( G ). EGFP-positive cells in upper layers were positive for NeuN (arrows), suggesting that they are neurons. Many EGFP-positive cells in lower layers were positive for Sox9 but negative for NeuN, Sox10 and Plp1 (arrowheads), suggesting that they are astrocytes. Many EGFP-positive cells in the white matter were positive for Sox10 and Plp1 (open arrowheads), suggesting that they are oligodendrocytes. ( H ) The percentages of EGFP-positive cells which were also NeuN-positive (neuron), Sox9-positive/Sox10-negative (astrocyte) or Plp1 -positive (oligodendrocyte). Unpaired Student’s t -test, * P < 0.05. Error bars represent mean ± SD. The statistical analyses were performed using Microsoft Excel ver. 16.43. (I) Three-dimensional reconstructed images of an EGFP-positive astrocyte (left) and an EGFP-positive oligodendrocyte (right). Scale bars: 200 μm ( C ), 30 μm ( D-G ). WM, white matter. Numbers indicate layers in the cerebral cortex.

Article Snippet: Antibodies used here were as follows: anti-NeuN antibody (Cell Signaling Technology), anti-Sox9 antibody (R&D systems), anti-Sox10 antibody (Santa Cruz Biotechnology), anti-Pax6 antibody (Millipore), anti-Tbr2 antibody (R&D systems), secondary antibodies conjugated with Alexa Fluor 647 (Molecular Probe), and secondary antibodies conjugated with Cy3 (Jackson ImmunoResearch).

Techniques: In Utero, Electroporation, Expressing, Transfection, Immunohistochemistry, In Situ Hybridization

Journal: Scientific Reports

Article Title: Glial cell type-specific gene expression in the mouse cerebrum using the piggyBac system and in utero electroporation

doi: 10.1038/s41598-021-84210-z

Figure Lengend Snippet: The Gfa2 promoter induced transgene expression in astrocytes specifically in the mouse cerebrum. ( A ) Schematics of pPB-Gfa2-EGFP and pCAG-PBase plasmids. pPB-Gfa2-EGFP drives EGFP expression under the Gfa2 promoter. ( B ) Experimental procedure. pPB-Gfa2-EGFP and pCAG-PBase were co-electroporated into the mouse cerebrum at E15.5, and coronal sections were prepared at P30. ( C ) Coronal sections of the electroporated cerebrum. Many EGFP-positive cells were distributed throughout the gray matter and the white matter. ( D – F ) Immunohistochemistry for NeuN ( D ), Sox9 and Sox10 ( E ), and in situ hybridization for Plp1 ( F ). Most EGFP-positive cells expressed Sox9 and were negative for NeuN, Sox10 and Plp1 (arrowheads). ( G ) The percentages of EGFP-positive cells which were also NeuN-positive (neuron), Sox9-positive/Sox10-negative (astrocyte) or Plp1 -positive (oligodendrocyte). The Gfa2 promoter drove EGFP expression in astrocytes selectively when combined with the piggyBac system and in utero electroporation. Unpaired Student’s t -test, * P < 0.0005. Error bars represent mean ± SD. The statistical analyses were performed using Microsoft Excel ver. 16.43. Scale bars: 200 μm ( C ), 30 μm ( D–F ). TR, terminal region; WM, white matter. Numbers indicate layers in the cerebral cortex.

Article Snippet: Antibodies used here were as follows: anti-NeuN antibody (Cell Signaling Technology), anti-Sox9 antibody (R&D systems), anti-Sox10 antibody (Santa Cruz Biotechnology), anti-Pax6 antibody (Millipore), anti-Tbr2 antibody (R&D systems), secondary antibodies conjugated with Alexa Fluor 647 (Molecular Probe), and secondary antibodies conjugated with Cy3 (Jackson ImmunoResearch).

Techniques: Expressing, Immunohistochemistry, In Situ Hybridization, In Utero, Electroporation

Journal: Scientific Reports

Article Title: Glial cell type-specific gene expression in the mouse cerebrum using the piggyBac system and in utero electroporation

doi: 10.1038/s41598-021-84210-z

Figure Lengend Snippet: A Blbp promoter did not induce astrocyte-specific transgene expression when used with the piggyBac system and in utero electroporation. ( A ) Schematics of pPB-Blbp-EGFP and pCAG-PBase plasmids. pPB-Blbp-EGFP drives EGFP expression under a Blbp promoter. ( B ) Experimental procedure. pPB-Blbp-EGFP and pCAG-PBase were co-electroporated into the mouse cerebrum at E15.5, and coronal sections were prepared at P30. ( C ) Coronal sections of the electroporated cerebrum. Many EGFP-positive cells were distributed throughout the gray matter and the white matter. ( D – G ) Immunohistochemistry for NeuN (D), Sox9 and Sox10 ( E ), and in situ hybridization for Plp1 ( F ). Although EGFP-positive cells contained Sox9-positive/Sox10-negative astrocytes (arrowheads), they also contained NeuN-positive neurons (arrows) and Plp1 -positive oligodendrocytes (open arrowheads). ( G ) The percentages of EGFP-positive cells which were also NeuN-positive (neuron), Sox9-positive/Sox10-negative (astrocyte) or Plp1 -positive (oligodendrocyte). This Blbp promoter drove gene expression not only in astrocytes but also in neurons and oligodendrocytes. Unpaired Student’s t -test, * P < 0.05. Error bars represent mean ± SD. The statistical analyses were performed using Microsoft Excel ver. 16.43. Scale bars: 200 μm ( C ), 30 μm ( D – F ). TR, terminal region; WM, white matter. Numbers indicate layers in the cerebral cortex.

Article Snippet: Antibodies used here were as follows: anti-NeuN antibody (Cell Signaling Technology), anti-Sox9 antibody (R&D systems), anti-Sox10 antibody (Santa Cruz Biotechnology), anti-Pax6 antibody (Millipore), anti-Tbr2 antibody (R&D systems), secondary antibodies conjugated with Alexa Fluor 647 (Molecular Probe), and secondary antibodies conjugated with Cy3 (Jackson ImmunoResearch).

Techniques: Expressing, In Utero, Electroporation, Immunohistochemistry, In Situ Hybridization, Gene Expression

Journal: Scientific Reports

Article Title: Glial cell type-specific gene expression in the mouse cerebrum using the piggyBac system and in utero electroporation

doi: 10.1038/s41598-021-84210-z

Figure Lengend Snippet: A Plp1 promoter induced transgene expression in oligodendrocytes specifically in the mouse cerebrum. ( A ) Schematics of pPB-Plp1-EGFP and pCAG-PBase plasmids. pPB-Plp1-EGFP drives EGFP expression under a Plp1 promoter. ( B ) Experimental procedure. pPB-Plp1-EGFP and pCAG-PBase were co-electroporated into the mouse cerebrum at E15.5, and coronal sections were prepared at P30. ( C ) Coronal sections of the electroporated cerebrum. Most EGFP-positive cells were distributed in the white matter. ( D–G ) Immunohistochemistry for NeuN ( D ), Sox9 and Sox10 ( E ), and in situ hybridization for Plp1 ( F ). Most EGFP-positive cells were positive for Plp1 and negative for NeuN and Sox9 (open arrowheads). ( G ) The percentages of EGFP-positive cells which are also NeuN-positive (neuron), Sox9-positive/Sox10-negative (astrocyte) or Plp1 -positive (oligodendrocyte). This Plp1 promoter drove gene expression in oligodendrocytes selectively when combined with the piggyBac system and in utero electroporation. Unpaired Student’s t -test, * P < 0.00005. Error bars represent mean ± SD. The statistical analyses were performed using Microsoft Excel ver. 16.43. Scale bars: 200 μm ( C ), 30 μm ( D-F ). TR, terminal region; WM, white matter. Numbers indicate layers in the cerebral cortex.

Article Snippet: Antibodies used here were as follows: anti-NeuN antibody (Cell Signaling Technology), anti-Sox9 antibody (R&D systems), anti-Sox10 antibody (Santa Cruz Biotechnology), anti-Pax6 antibody (Millipore), anti-Tbr2 antibody (R&D systems), secondary antibodies conjugated with Alexa Fluor 647 (Molecular Probe), and secondary antibodies conjugated with Cy3 (Jackson ImmunoResearch).

Techniques: Expressing, Immunohistochemistry, In Situ Hybridization, Gene Expression, In Utero, Electroporation

Journal: Scientific Reports

Article Title: Glial cell type-specific gene expression in the mouse cerebrum using the piggyBac system and in utero electroporation

doi: 10.1038/s41598-021-84210-z

Figure Lengend Snippet: An Mbp promoter induced transgene expression into oligodendrocytes specifically in the mouse cerebrum. ( A ) Schematic of pPB-Mbp-EGFP and pCAG-PBase plasmid. pPB-Mbp-EGFP drives EGFP expression under an Mbp promoter. ( B ) Experimental procedure. pPB-Mbp-EGFP and pCAG-PBase were co-electroporated into the mouse cerebrum at E15.5, and coronal sections were prepared at P30. ( C ) Coronal sections of the electroporated cerebrum. Most EGFP-positive cells were accumulated in the white matter. ( D–G ) Immunohistochemistry for NeuN ( D ), Sox9 and Sox10 ( E ), and in situ hybridization for Plp1 ( F ). Most EGFP-positive cells were positive for Plp1 and negative for NeuN and Sox9 (open arrowheads). ( G ) The percentages of EGFP-positive cells which were also NeuN-positive (neuron), Sox9-positive/Sox10-negative (astrocyte) or Plp1 -positive (oligodendrocyte). This Mbp promoter drove gene expression in oligodendrocytes selectively when combined with the piggyBac system and in utero electroporation. Unpaired Student’s t -test, * P < 0.00005. Error bars represent mean ± SD. The statistical analyses were performed using Microsoft Excel ver. 16.43. Scale bars: 200 μm ( C ), 30 μm ( D-F ). TR, terminal region; WM, white matter. Numbers indicate layers in the cerebral cortex.

Article Snippet: Antibodies used here were as follows: anti-NeuN antibody (Cell Signaling Technology), anti-Sox9 antibody (R&D systems), anti-Sox10 antibody (Santa Cruz Biotechnology), anti-Pax6 antibody (Millipore), anti-Tbr2 antibody (R&D systems), secondary antibodies conjugated with Alexa Fluor 647 (Molecular Probe), and secondary antibodies conjugated with Cy3 (Jackson ImmunoResearch).

Techniques: Expressing, Plasmid Preparation, Immunohistochemistry, In Situ Hybridization, Gene Expression, In Utero, Electroporation

Journal: JOR Spine

Article Title: An Ex Vivo Model of Intervertebral Disc Degeneration for Assessing Retention of Injectable Cell‐Based Grafts

doi: 10.1002/jsp2.70144

Figure Lengend Snippet: Immunostaining of human NCS retained in bovine IDD model. An expression of (A) aggrecan, (B) SOX‐9, and (C) cleaved caspase 3 (cCas3) in mCherry‐transduced NCS at day 1 and 7 post‐injection ( n = 3 animals per group). (D) Semi‐quantification of target‐positive cells (%), expressed relative to the total number of cells (DAPI+/mCherry+) within each ROI in a representative histological section. Scale bar: 400 μm.

Article Snippet: Sections were subjected to enzymatic epitope retrieval and blocked with 1% bovine serum albumin (BSA) (A9647, Sigma) supplemented with triton X‐100 (1:1000, 93 418, Sigma), followed by application of primary antibodies, namely anti‐cleaved caspase 3 (1:300, polyclonal, 9661, Cell Signaling; immunoreactive in bovine and human cells), mCherry (1:1500, Rb pAB, AB167453 , Abcam), anti‐Aggrecan [6‐B‐4] (1:200, ab3778, Abcam; immunoreactive in human cells), SOX9 (1:100, Rb pAB, NBP2‐24659, Novus Biologicals; immunoreactive in human and bovine cells), MMP13 (1:100, 18 165‐1‐AP, Proteintech; immunoreactive in human cells), COX2 (1:100, MA5‐14568, Thermofisher; immunoreactive in human and bovine cells).

Techniques: Immunostaining, Expressing, Injection