Journal: Circulation Research

Article Title: Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture

doi: 10.1161/CIRCRESAHA.120.317295

Figure Lengend Snippet: Protein interaction network analysis reveals numerous interactions of proteins that are differentially abundant in aortas of SR-uPA +/0 mice. A protein-protein relational network was built based on experimentally validated direct interactions. The network is comprised of 87 proteins, each portrayed as a circular node (all nodes are identified in Data Set V in the Data Supplement ). Key highly connected nodes (hubs) are labeled together with 2 members of the matrix metalloproteinase family of extracellular proteases and several extracellular matrix components. ACTB indicates beta actin; AGRN, agrin; BCAM, basal cell adhesion molecule; ELN, elastin; FBLN5, fibulin 5; FN1, fibronectin 1; HSPG2, heparan sulfate proteoglycan 2; LAMA5, laminin subunit alpha 5; LAMB2, laminin subunit beta 2; LAMC1, laminin subunit gamma 1; LTBP4, latent transforming growth factor binding protein 4; MMP2, matrix metalloproteinase 2; MMP3, matrix metalloproteinase 3; MYH9, myosin heavy chain 9; NID1, nidogen1; NID2, nidogen 2; and PLAU, urokinase-type plasminogen activator.

Article Snippet: Primary antibodies and dilutions were rabbit anti-mouse laminin α5 serum (clone 504; a gift from Dr Lydia Sorokin, Munster University; 1:1000), mouse monoclonal anti-mouse endostatin (a fragment of COL18A1; Santa Cruz Biotechnology sc-32720; 1:100), and rat monoclonal anti-HSPG2 (Abcam ab2501; 1:100).

Techniques: Labeling, Binding Assay

Journal: Circulation Research

Article Title: Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture

doi: 10.1161/CIRCRESAHA.120.317295

Figure Lengend Snippet: Representative peptographs of extracts of ruptured vs stable human plaque segments. A and B , Extracts of ruptured (red) and adjacent stable (blue) segments of 5 human carotid plaques were analyzed using the PROTOMAP protocol. The extracts were subjected to SDS-PAGE, and the gels were cut into 22 slices, each corresponding to a molecular weight range. After in-gel trypsin digestion, peptides were extracted, identified by tandem mass spectrometry, and spectral counts were aggregated over all 22 slices. A , Proteins with differential abundance of lower-molecular weight peptides in extracts of ruptured vs. stable segments: ceruloplasmin (CP), angiotensinogen (AGT), ITIH4 (inter-alpha-trypsin inhibitor heavy chain), GPLD1 (phosphatidylinositol-glycan-specific phospholipase D), FA5 (coagulation factor V), SERPIND1 (serpin family D member 1), LAMB2 (laminin subunit beta-2), and SVIL (supervillin). B , ECM (Extracellular matrix) proteins that are significantly less abundant in extracts of ruptured vs stable human plaque segments: ACAN (aggrecan core protein), FBLN5 (fibulin 5), FMOD (fibromodulin), HAPLN (hyaluronan and proteoglycan link protein 1), LAMA5 (laminin subunit alpha 5), MFAP4 (microfibril-associated glycoprotein 4), LTBP1 (latent-transforming growth factor beta-binding protein 1), and VCAN (versican). A and B , Horizontal bars in each peptograph portray the total spectral counts for protein-specific peptides in each of the 22 gel slices (mean±SEM; n=5). Gel-slice number is on the leftward y -axis; molecular weight of the gel slices (in kilodaltons) is on the rightward y -axis.

Article Snippet: Primary antibodies and dilutions were rabbit anti-mouse laminin α5 serum (clone 504; a gift from Dr Lydia Sorokin, Munster University; 1:1000), mouse monoclonal anti-mouse endostatin (a fragment of COL18A1; Santa Cruz Biotechnology sc-32720; 1:100), and rat monoclonal anti-HSPG2 (Abcam ab2501; 1:100).

Techniques: SDS Page, Molecular Weight, Mass Spectrometry, Glycoproteomics, Coagulation, Binding Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Myocilin Regulates Metalloprotease 2 Activity Through Interaction With TIMP3

doi: 10.1167/iovs.16-20336

Figure Lengend Snippet: TIMP3 is identified as a myocilin-binding protein. (A) Western blot analysis of immunoprecipitates with anti-myocilin antibodies or rabbit IgG from eye lysates of Myoc null (KO), WT, and Tg mice expressing WT (wt MYOC) or mutated mouse myocilin (m MYOC). The efficiency and specificity of immunoprecipitation were confirmed using anti-myocilin antibodies. The peptide number (N) indicates the number of peptides associated with myocilin (MYOC) or TIMP3 identified in the immunoprecipitates by shotgun proteomics. (B) Myocilin interacts with TIMP3 in HEK293 cells. HEK293 cells were cotransfected with indicated HA-tagged human TIMP constructs and a FLAG-tagged human myocilin construct. The lysates were immunoprecipitated with anti-HA antibodies and the precipitates were probed with anti-FLAG antibodies. (C) Myocilin interacts with TIMP3 in HepG2 cells. HepG2 cells were cotransfected with a myc-tagged TIMP3 construct and indicated FLAG-tagged myocilin constructs. The lysates were immunoprecipitated with anti-FLAG antibodies and the precipitates were probed with anti-myc antibodies. The arrowhead marks TIMP3 band and the arrow marks immunoglobulin band. (D) Model of TIMP3–OLF interaction based on computational docking, suggesting that OLF does not compete for the MMP binding site on TIMP3, which is near its N-terminus.

Article Snippet: Sections were fixed with 4% PFA for 10 minutes on ice, blocked with Blocker Casein PBS (Thermo Fisher) for 30 minutes at room temperature, and then incubated with primary antibodies against mouse myocilin or TIMP3 (Novus Biologicals) at 4°C overnight.

Techniques: Binding Assay, Western Blot, Expressing, Immunoprecipitation, Construct

Journal: Investigative Ophthalmology & Visual Science

Article Title: Myocilin Regulates Metalloprotease 2 Activity Through Interaction With TIMP3

doi: 10.1167/iovs.16-20336

Figure Lengend Snippet: Distribution of myocilin and TIMP3 in the ocular tissues. Cryosections of the FVRD mouse eyes were stained with anti-myocilin and TIMP3 antibodies. (A–C, G–I) Eye drainage structures: (A) Myocilin is detected mainly in the TM. (B) The image of the same area as in (A) obtained using DIC microscopy for better visualization of the tissue orientation. (C) Enlarged image of the TM area boxed in (A). (G) TIMP3 is detected in the TM and ciliary body. (H) The image of the same area as in (D) obtained using DIC microscopy. (H) Enlarged image of the TM area boxed in (G). (D–F, J–L) Posterior part of the eye: (D) Myocilin was detected in the choroid and sclera. (E) The image of the same area as in (D) obtained using DIC microscopy. (F) Enlarged image of the RPE–sclera region. (J) TIMP3 was detected in the RPE, Bruch's membrane, choroid, and sclera. (K) The image of the same area as in (J) obtained using DIC microscopy. (L) An enlarged image of RPE–sclera region. Abbreviations are as in . BrM, Bruch's membrane.

Article Snippet: Sections were fixed with 4% PFA for 10 minutes on ice, blocked with Blocker Casein PBS (Thermo Fisher) for 30 minutes at room temperature, and then incubated with primary antibodies against mouse myocilin or TIMP3 (Novus Biologicals) at 4°C overnight.

Techniques: Staining, Microscopy, Membrane

Journal: Investigative Ophthalmology & Visual Science

Article Title: Myocilin Regulates Metalloprotease 2 Activity Through Interaction With TIMP3

doi: 10.1167/iovs.16-20336

Figure Lengend Snippet: Myocilin enhances the inhibitory activity of TIMP3 toward MMP2. (A) The purity of TIMP3 and myocilin was estimated by SDS-PAGE. (B) MMP2 (50 nM) was coincubated with indicated different concentrations of TIMP3. The fluorescence signals representing the protease activity of MMP2 were monitored for 2 hours. Arrow indicates TIMP3 concentration selected for further studies. (C) Myocilin (200 nM) or control IgG (200 nM) was preincubated with TIMP3 (100 nM). TIMP3 alone or the protein mixtures were added to the MMP2 proteolytic reaction. Error bars represent ±SD of triplicate reactions. Statistically significant differences between two groups (+ MMP2, TIMP3 and + MMP2, TIMP3, MYOC) at different time points are indicated by asterisks (P < 0.05).

Article Snippet: Sections were fixed with 4% PFA for 10 minutes on ice, blocked with Blocker Casein PBS (Thermo Fisher) for 30 minutes at room temperature, and then incubated with primary antibodies against mouse myocilin or TIMP3 (Novus Biologicals) at 4°C overnight.

Techniques: Activity Assay, SDS Page, Fluorescence, Concentration Assay, Control

Journal: European journal of orthodontics

Article Title: Proinflammatory mediators related to orthodontically induced periapical root resorption in rat mandibular molars.

doi: 10.1093/ejo/cjx033

Figure Lengend Snippet: Figure 2. Expression of monocyte and osteoclast markers, and proinfl ammatory mediators in orthodontically induced root resorption and physiological tooth drift areas. Cells positive for NSE (non-specific esterase), TRAP (tartrate-resistant acid phosphatase), IL-1α, IL-1β, TNF-α, cyclooxygenases COX-1 and COX-2, and PGE2 (prostaglandin E2) were detected by histochemistry and quantified as the total cell number per unit area using Image-Pro. Drift, untreated control (n = 4); 8 and 15 days, root resorption areas after 8 and 15 days, respectively, of mesio-occlusal tooth movement (n = 8). *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the drift group. Horizontal lines of the box and whisker plot diagram represent 95th, 75th, 50th, 25th, and 5th percentiles from the top.

Article Snippet: Sections were then pre-treated with 2% normal goat or rabbit serum (NSS) depending on the origin of the primary antibody (Vector Laboratories) in PBST for 30 min, and incubated with the primary antibodies against rat IL-1α and IL-1β (both rabbit polyclonal, 1:300; Endogen, Woburn, Massachusetts, USA), rat TNF-α (goat polyclonal, 1:1,000; R&D Systems, Inc., Minneapolis, Minnesota, USA), murine COX-1 (1:300) and COX-2 (1:100) (both rabbit polyclonal; Cayman Chemical Company, Ann Arbor, Michigan, USA), and rat PGE2 (rabbit polyclonal, 1:500; Assay Designs, Inc., Ann Arbor, Michigan, USA) in 2% NSS for 20 h at 4°C (21, 22).

Techniques: Expressing, Control, Whisker Assay

Journal: European journal of orthodontics

Article Title: Proinflammatory mediators related to orthodontically induced periapical root resorption in rat mandibular molars.

doi: 10.1093/ejo/cjx033

Figure Lengend Snippet: Figure 1. Localization of monocyte and osteoclast markers, and proinfl ammatory mediators in orthodontically induced root resorption areas. Eight- week-old rats were observed for physiological distal tooth drift (control, A) or subjected to mesio-occlusal tooth movement for 8 (B) and 15 (C) days and analyzed for the expression of indicated factors by histochemistry. (D) Inflammation site (positive control staining). NSE, non-specific esterase (black staining); TRAP, tartrate-resistant acid phosphatase (red staining); IL-1α, interleukin-1α; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; COX-1, cyclooxygenase 1; COX-2, cyclooxygenase 2; PGE2, prostaglandin E2; B, alveolar bone; T, tooth; Arrows, COX-1 positive cells in control; Scale bar, 50 μm; In the last row, a schema of the observation area and low magnification images by hematoxylin and eosin (HE) staining of root resorption after 8 (column B) and 15 (C) days; M, mesial side; Arrow heads, root resorption area; Scale bar, 250 μm.

Article Snippet: Sections were then pre-treated with 2% normal goat or rabbit serum (NSS) depending on the origin of the primary antibody (Vector Laboratories) in PBST for 30 min, and incubated with the primary antibodies against rat IL-1α and IL-1β (both rabbit polyclonal, 1:300; Endogen, Woburn, Massachusetts, USA), rat TNF-α (goat polyclonal, 1:1,000; R&D Systems, Inc., Minneapolis, Minnesota, USA), murine COX-1 (1:300) and COX-2 (1:100) (both rabbit polyclonal; Cayman Chemical Company, Ann Arbor, Michigan, USA), and rat PGE2 (rabbit polyclonal, 1:500; Assay Designs, Inc., Ann Arbor, Michigan, USA) in 2% NSS for 20 h at 4°C (21, 22).

Techniques: Control, Expressing, Positive Control, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells

doi: 10.3389/fncel.2019.00420

Figure Lengend Snippet: Dynamic changes in Slit1 expression in DRG after sciatic nerve injury. The expression of Slit1 in DRG was detected by immunofluorescence histochemistry and western blotting at different time points after SNC. (A–E) Contralateral DRG sections. (F–J) Ipsilateral DRG sections. The white arrows indicate strongly Slit1-positive neurons, and the green arrow indicates weakly Slit1-positive neurons. Scale bar = 100 μm. (K) At different time points, the numbers of strongly Slit1-positive DRG neurons on both sides were compared, and one-way repeated measures ANOVA and paired t -tests were used for statistical analysis. “ ∗ ” in comparison with the control side, the difference is statistically significant at P < 0.05; “#” in comparisons of the groups at 1, 3 or 7 days after injury with the group at 14 days after injury, the difference is statistically at P < 0.01. (L) Detection of DRG Slit1 protein expression at different time points by western blotting ( N = 3). The level of β-actin was detected as loading control. (M) At different time points, the relative gray values of DRG Slit1 expression on the two sides were compared. N = 3, and one-way repeated measures ANOVA and paired t -tests were used for statistical analysis, “ ∗ ” compared to the contralateral side, P < 0.01. CON: contralateral DRG, IPS: ipsilateral (crush side) DRG.

Article Snippet: However, we used a mouse polyclonal primary antibody against Slit1 (ab115892, immunogen: Synthetic peptide corresponding to mouse Slit1 aa 497–504 conjugated to keyhole limpet hemocyanin), which was predicted to be able to react with rat Slit1.

Techniques: Expressing, Immunofluorescence, Western Blot, Comparison, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells

doi: 10.3389/fncel.2019.00420

Figure Lengend Snippet: Slit1 expression in SGCs of injured DRG. Slit1 expression in the SGCs of injured DRG was detected by immunofluorescence histochemistry. (A) Slit1 (red) immunofluorescence staining of contralateral DRG sections randomly selected from the different groups. (B–F) Slit1 immunofluorescence staining of ipsilateral DRG at different time points after operation. The arrow indicates Slit1-positive SGCs. (G) GS (green) immunofluorescence staining of DRG sections on the 7th day after sciatic nerve crush. (H) Merged images from panels (D,E) . (I) Summary histograms depicting the changes in the Slit1 RFI in SGCs (one-way repeated measures ANOVA, ∗ P < 0.01); RFI, relative fluorescence intensity. (J) Changes in the number of strongly Slit1-positive DRG neurons and the RFI of Slit1 expression in SGCs at different time points after injury are shown. Scale bar = 100 μm.

Article Snippet: However, we used a mouse polyclonal primary antibody against Slit1 (ab115892, immunogen: Synthetic peptide corresponding to mouse Slit1 aa 497–504 conjugated to keyhole limpet hemocyanin), which was predicted to be able to react with rat Slit1.

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells

doi: 10.3389/fncel.2019.00420

Figure Lengend Snippet: Effects of DRG-injured neuronal soma on Slit1 expression in SGCs. The Slit1 and ATF3 double immunofluorescence labeling method and the Slit1 and FG retrograde labeling method were used to show the expression of Slit1 in SGCs around normal and injured neurons. (A–C) Double immunofluorescence labeling of Slit1 and ATF3 was carried out on DRG sections from 7 days after sciatic nerve crush. (A) Slit1 (red) immunofluorescence staining. (B) ATF3 (green) immunofluorescence staining. (C) Merged images of panels (A,B) . The thick arrow indicates ATF3-positive neurons, and the thin arrow indicates ATF3-negative neurons, scale bar = 25 μm. (D–F) Slit1 immunofluorescence staining and FG retrograde labeling. (D) DRG Slit1 immunofluorescence on the 7th day after the operation. (E) FG (white)-traced positive neurons. (F) Merged images of panels (D,E) . Scale bar = 200 μm. The large framed image is an enlargement of the small frame, and thin arrows indicate FG-negative neurons in panels (F) . (G) Summary histograms depict the changes in the RFI of Slit1 in SGCs surrounding ATF3-positive and negative neurons determined with paired t -tests, ∗ P < 0.001; bars represent the standard error of the mean; RFI, relative fluorescence intensity. (H) Summary histograms depict the changes in the RFI of Slit1 expression in SGCs surrounding FG-negative and FG-positive neurons determined with paired t -tests, ∗ P < 0.001; bars represent the standard error of the mean; RFI, relative fluorescence intensity.

Article Snippet: However, we used a mouse polyclonal primary antibody against Slit1 (ab115892, immunogen: Synthetic peptide corresponding to mouse Slit1 aa 497–504 conjugated to keyhole limpet hemocyanin), which was predicted to be able to react with rat Slit1.

Techniques: Expressing, Immunofluorescence, Labeling, Staining, Fluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells

doi: 10.3389/fncel.2019.00420

Figure Lengend Snippet: Expression of Slit1 in SGCs directly in contact with the neuronal soma in vitro (A–C) Primary cultured DRG cells were double labeled with Slit1 and MAP2 immunochemistry, nuclear staining by DAPI. (A) Slit1 (red) and DAPI (blue) merged image. The box 1 shows SGCs in contact with neuron bodies, Panel (A1) is its enlarged image, with arrows indicating SGCs in contact with neuronal soma (white asterisks); and the box 2 shows SGCs located far from neuron bodies as showed in its enlarged image (A2) . (B) MAP2 (green) image. (C) Slit1 (red), MAP2 (green) and DAPI (blue) merged image. (D) Slit1 and GS double immunostaining were performed on cultured DRG cells, nuclear staining by DAPI. The box 1 shows SGCs in contact with Slit1-positive neuronal soma (white asterisks). The box 2 shows SGCs located far from neuron bodies. Panels (D1,D2) lines of pictures were local enlarged images of the box 1 and box 2 showed in panel (D) , respectively. The white asterisk indicates the neuronal soma. Slit1 (red), MAP2 (green) and DAPI (blue) were showed in panels (D,D1,D2) . (E) Summary histograms depict the changes in Slit1 expression in two different populations of SGCs, ∗ shows the expression of Slit1 in SGCs of population 1 compared with that in SGCs of population 2 analyzed with paired t -tests, ∗ P < 0.001; bars represent the standard error of the mean; RFI, relative fluorescence intensity. Scale bar = 50 μm was showed in panels (A–C) ; Scale bar = 15 μm was showed in panel (D) .

Article Snippet: However, we used a mouse polyclonal primary antibody against Slit1 (ab115892, immunogen: Synthetic peptide corresponding to mouse Slit1 aa 497–504 conjugated to keyhole limpet hemocyanin), which was predicted to be able to react with rat Slit1.

Techniques: Expressing, In Vitro, Cell Culture, Labeling, Staining, Double Immunostaining, Fluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells

doi: 10.3389/fncel.2019.00420

Figure Lengend Snippet: Slit1 expression in SGCs decreased after inhibition of the P2X7 receptor with BBG. After right sciatic nerve crush, BBG (P2X7 receptor inhibitor) was injected intraperitoneally. The expression of Slit1 on the 7th day DRG was detected by double immunofluorescence staining and western blotting. (A–D) Slit1 immunofluorescence staining (red). (E–H) Merged images of Slit1 (red) and GS (green) immunofluorescence staining. Scale bar = 200 μm. (I) the level of Slit1 protein was detected by western blotting ( N = 3), the level of β-actin was detected as loading control. (J) The level of Slit1 before and after BBG treatment on the 7th day post-SNC DRG was analyzed with the paired t -test. “ ∗ ” compared with the control side, the level of Slit1 in IPS side was upregulated, P < 0.001; “#” compared with the control side and the ipsilateral side before BBG treatment respectively, the level of Slit1 in “IPS + BBG” was downregulated, P < 0.001; The change was not significant in “CON + BBG”, P > 0.05. “ ∗∗ ” compared with “CON + BBG” group, the level of Slit1 in “IPS + BBG” was much lower than that in “CON + BBG” group, P < 0.001. CON: contralateral DRG, IPS: ipsilateral DRG.

Article Snippet: However, we used a mouse polyclonal primary antibody against Slit1 (ab115892, immunogen: Synthetic peptide corresponding to mouse Slit1 aa 497–504 conjugated to keyhole limpet hemocyanin), which was predicted to be able to react with rat Slit1.

Techniques: Expressing, Inhibition, Injection, Double Immunofluorescence Staining, Western Blot, Immunofluorescence, Staining, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells

doi: 10.3389/fncel.2019.00420

Figure Lengend Snippet: Schematic diagram of the role of Slit1 in the communication between neurons and SGCs. After peripheral nerve injury, neurons activate perineuronal SGCs, leading to upregulation of Slit1 expression through the VNUT-ATP-P2X7R pathway, and SGCs then promote the regeneration of injured neurons through the Slit1-Robo2-srGAP3 pathway ( ; ).

Article Snippet: However, we used a mouse polyclonal primary antibody against Slit1 (ab115892, immunogen: Synthetic peptide corresponding to mouse Slit1 aa 497–504 conjugated to keyhole limpet hemocyanin), which was predicted to be able to react with rat Slit1.

Techniques: Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells

doi: 10.3389/fncel.2019.00420

Figure Lengend Snippet: Primary and secondary antibodies used in this study.

Article Snippet: However, we used a mouse polyclonal primary antibody against Slit1 (ab115892, immunogen: Synthetic peptide corresponding to mouse Slit1 aa 497–504 conjugated to keyhole limpet hemocyanin), which was predicted to be able to react with rat Slit1.

Techniques: Control

Journal: Cell death & disease

Article Title: Inverse and reciprocal regulation of p53/p21 and Bmi-1 modulates vasculogenic differentiation of dental pulp stem cells.

doi: 10.1038/s41419-021-03925-z

Figure Lengend Snippet: Fig. 1 Characterization of MSC and endothelial markers in dental pulp stem cells. Cell surface markers detected by flow cytometry in DPSC, SHED, and HDMEC under standard culture conditions for each cell type. a Flow plots depicting expression of MSC markers (i.e., CD73, CD90, CD105, CD34, CD44) using IgG-APC as isotype-matched control. b Flow plots depicting expression of endothelial cell-related proteins (VEGFR1 and CD146) using IgG-PE and IgG-FITC were used as isotype-matched controls. c RT-PCR for odontogenic/osteogenic markers (DSPP, DMP-1), endothelial cell-related markers (VEGFR1, VEGFR2, CD146, CD31, VE-cadherin) in primary human odontoblasts, dental pulp tissue, DPSC, SHED, or HDMEC. GAPDH was used as a loading control.

Article Snippet: Membranes were blocked with 5% non-fat milk in 1× TBS containing 0.3% Tween-20, then incubated with the following primary antibodies overnight at 4°C: rabbit anti-human VEGFR2 (SC-504), c-Jun (SC-1694); mouse anti-human VE-cadherin (SC-9989), CD31 (SC-365804), CD146 (SC-18837), MDM2 (SC965), p53 (SC-126), phosphor-c-Jun (SC-822), phosphor-JNK (SC-6254), JNK (SC-1648), mouse anti-human β-actin conjugated with HRP (SC-47778HRP) (Santa Cruz Biotechnology; Santa Cruz, CA, USA); mouse anti-GAPDH (MAB374; Millipore Sigma); rabbit anti-human Tie-2 (7403), Bmi-1(6964), PDGFR-a (5241), PDGFR-β (4564), p21 (2947) (Cell Signaling; Danvers, MA, USA); mouse anti-human SMA-alpha; CBL171; Millipore Sigma).

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

Journal: Cell death & disease

Article Title: Inverse and reciprocal regulation of p53/p21 and Bmi-1 modulates vasculogenic differentiation of dental pulp stem cells.

doi: 10.1038/s41419-021-03925-z

Figure Lengend Snippet: Fig. 3 Stabilization of p53 by blockade of the p53-MDM2 binding inhibits vasculogenic differentiation of dental pulp stem cells. a shRNA- p53 transduced DPSC were treated with 0–2.5 µM MI-773 and 0–10 µM APG-115 for 24 h, western blots were performed for P53, MDM2, and p21, β-actin was used as loading control. b DPSC were cultured with endothelial differentiation medium in the presence of 50 ng/ml VEGF and 0–0.25 µM MI-773 for 14 days, western blots for p53, MDM2, p21, Bmi-1, VEGFR2, Tie-2, and CD31. The density of protein expression was normalized with β-actin. c, d 1 × 104 DPSC were seeded in growth factor-reduced matrigel and cultured in endothelial differentiation medium (EGM2) in the presence of 0–0.5 µM MI-773 for 13 days. c photographs of sprout, scale bar:100 µm. d Graph depicting the number of sprouts formed in c. Asterisk indicates p < 0.001, as determined by one-way ANOVA followed by a post hoc test (Tukey’s test).

Article Snippet: Membranes were blocked with 5% non-fat milk in 1× TBS containing 0.3% Tween-20, then incubated with the following primary antibodies overnight at 4°C: rabbit anti-human VEGFR2 (SC-504), c-Jun (SC-1694); mouse anti-human VE-cadherin (SC-9989), CD31 (SC-365804), CD146 (SC-18837), MDM2 (SC965), p53 (SC-126), phosphor-c-Jun (SC-822), phosphor-JNK (SC-6254), JNK (SC-1648), mouse anti-human β-actin conjugated with HRP (SC-47778HRP) (Santa Cruz Biotechnology; Santa Cruz, CA, USA); mouse anti-GAPDH (MAB374; Millipore Sigma); rabbit anti-human Tie-2 (7403), Bmi-1(6964), PDGFR-a (5241), PDGFR-β (4564), p21 (2947) (Cell Signaling; Danvers, MA, USA); mouse anti-human SMA-alpha; CBL171; Millipore Sigma).

Techniques: Binding Assay, shRNA, Western Blot, Control, Cell Culture, Expressing

Journal: Cell death & disease

Article Title: Inverse and reciprocal regulation of p53/p21 and Bmi-1 modulates vasculogenic differentiation of dental pulp stem cells.

doi: 10.1038/s41419-021-03925-z

Figure Lengend Snippet: Fig. 5 p53-dependent vasculogenic differentiation requires inverse and reciprocal regulation of p21 and Bmi-1. a–d Western blot for p53, MDM2, p21, and Bmi-1 in dental pulp cells. a Untransduced and shRNA-transduced DPSC. b–d Dental pulp cells were treated with 0–10 µM MI-773 (b), 0–10 µM APG-115 (c), and 0–10 µM Bmi-1 inhibitor (PTC-209) (d) for 24 h. e SHED were cultured with 5% FBS–MEM in the presence of 50 ng/ml VEGF with or without 0–2.5 µM PTC-209 for 14 days, western blots for VEGFR2, Tie-2, Bmi-1, and p21, GAPDH was used as a loading control. The density of protein expression was normalized with GAPDH. f, g 1 × 104 SHED were seeded in growth factor-reduced matrigel and cultured with EGM2 in the presence of 0–2.5 µM PTC-209 for different time points. f photographs of sprout in SHED. Scale bar: 100 µm. g Graph depicting the number of sprouts formed in f, Asterisk indicates p < 0.001, as determined by one-way ANOVA followed by a post hoc test (Tukey’s test).

Article Snippet: Membranes were blocked with 5% non-fat milk in 1× TBS containing 0.3% Tween-20, then incubated with the following primary antibodies overnight at 4°C: rabbit anti-human VEGFR2 (SC-504), c-Jun (SC-1694); mouse anti-human VE-cadherin (SC-9989), CD31 (SC-365804), CD146 (SC-18837), MDM2 (SC965), p53 (SC-126), phosphor-c-Jun (SC-822), phosphor-JNK (SC-6254), JNK (SC-1648), mouse anti-human β-actin conjugated with HRP (SC-47778HRP) (Santa Cruz Biotechnology; Santa Cruz, CA, USA); mouse anti-GAPDH (MAB374; Millipore Sigma); rabbit anti-human Tie-2 (7403), Bmi-1(6964), PDGFR-a (5241), PDGFR-β (4564), p21 (2947) (Cell Signaling; Danvers, MA, USA); mouse anti-human SMA-alpha; CBL171; Millipore Sigma).

Techniques: Western Blot, shRNA, Cell Culture, Control, Expressing