Journal: The Journal of investigative dermatology

Article Title: Tumor Endothelial Marker 1 (TEM1/Endosialin/CD248) Enhances Wound Healing by Interacting with Platelet-Derived Growth Factor Receptors.

doi: 10.1016/j.jid.2019.03.1149

Figure Lengend Snippet: Figure 1. Expression of TEM1 in cutaneous wounds. (a) The tissue extracts harvested from the wound region at the indicated days after wounding were homogenized and analyzed for TEM1 expression by western blotting (n ¼ 3). The TEM1/GAPDH ratio was calculated based on the results of western blotting. (b) The tissue sections of the wounded skin were immunochemically stained for TEM1 protein on the indicated days after wounding (n ¼ 5). The nucleus was stained with hematoxylin. The integrated density of TEM1 staining (brown) for each field in the wound bed was determined using the ImageJ software. The relative intensity at the indicated times was determined with reference to the unwounded samples (day 0). **P < 0.01 and ***P < 0.001. P-values were determined by one-way ANOVA. ANOVA, analysis of variance; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TEM1, tumor endothelium marker 1. Scale bar ¼ 100 mm.

Article Snippet: Cell lysates (1,000 mg) were incubated with 1 mg anti-TEM1 antibody (18160-1-AP, Proteintech) or normal rabbit IgG (sc-2027, Santa Cruz Biotechnology) and 20 ml of protein G-agarose beads (MilliporeSigma, Temecula, CA) Journal of Investigative Dermatology (2019), Volume - at 4 C overnight.

Techniques: Expressing, Western Blot, Staining, Software, Marker

Journal: The Journal of investigative dermatology

Article Title: Tumor Endothelial Marker 1 (TEM1/Endosialin/CD248) Enhances Wound Healing by Interacting with Platelet-Derived Growth Factor Receptors.

doi: 10.1016/j.jid.2019.03.1149

Figure Lengend Snippet: Figure 2. Delayed wound healing in TEM1 knockout mice. (a) Representative pictures depict the wound at the day after wounding in TEM1WT/WT and TEM1lacZ/lacZ

Article Snippet: Cell lysates (1,000 mg) were incubated with 1 mg anti-TEM1 antibody (18160-1-AP, Proteintech) or normal rabbit IgG (sc-2027, Santa Cruz Biotechnology) and 20 ml of protein G-agarose beads (MilliporeSigma, Temecula, CA) Journal of Investigative Dermatology (2019), Volume - at 4 C overnight.

Techniques: Knock-Out

Journal: The Journal of investigative dermatology

Article Title: Tumor Endothelial Marker 1 (TEM1/Endosialin/CD248) Enhances Wound Healing by Interacting with Platelet-Derived Growth Factor Receptors.

doi: 10.1016/j.jid.2019.03.1149

Figure Lengend Snippet: Figure 3. Reduced activation, proliferation, and collagen deposition of fibroblasts in the granulation tissues of wound in TEM1 knockout mice. (a) The tissue sections of the skin harvested from the wound region at day 14 were immunostained for a-SMA (green) and PCNA (red) (n ¼ 3), as markers for fibroblast activation and proliferation. The nucleus was stained with DAPI. a-SMAepositive cells relative to DAPI (upper panel), PCNA-positive cells relative to DAPI (middle panel), and double-positive cells relative to a-SMAepositive cells (lower panel) were quantified by the ImageJ software. Scale bar ¼ 100 mm. (b) Collagen deposition in the granulation tissues at day 14 after wound excision was detected by Masson’s trichrome staining. The percentage of collagen staining area (blue color) in the wound bed was determined using the ImageJ software (n ¼ 4). *P < 0.05 and ***P < 0.001. P-values were determined by unpaired two- tailed Student t test. a-SMA, a-smooth muscle actin; PCNA, proliferating cell nuclear antigen; TEM1, tumor endothelium marker 1. Scale bar ¼ 1 mm.

Article Snippet: Cell lysates (1,000 mg) were incubated with 1 mg anti-TEM1 antibody (18160-1-AP, Proteintech) or normal rabbit IgG (sc-2027, Santa Cruz Biotechnology) and 20 ml of protein G-agarose beads (MilliporeSigma, Temecula, CA) Journal of Investigative Dermatology (2019), Volume - at 4 C overnight.

Techniques: Activation Assay, Knock-Out, Staining, Software, Two Tailed Test, Marker

Journal: The Journal of investigative dermatology

Article Title: Tumor Endothelial Marker 1 (TEM1/Endosialin/CD248) Enhances Wound Healing by Interacting with Platelet-Derived Growth Factor Receptors.

doi: 10.1016/j.jid.2019.03.1149

Figure Lengend Snippet: Figure 4. The effect of TEM1 silencing on the migration, adhesion, and proliferation of fibroblasts. (a) The stable TEM1-knockdown fibroblasts were established by transfection with a lentivirus carrying TEM1 short hairpin RNA (shTEM1). The expression levels of TEM1 mRNA in the transfected fibroblasts were measured with RT-PCR. (b) Cell migration ability of the transfected fibroblasts was measured with scratch wound healing assay and (c) Transwell migration assay with 2% FBS as chemoattractant. Scale bar ¼ 100 mm. (d) The effect of TEM1 silencing on cell adhesion to the collagen- or fibronectin-coated 96-well plates was measured. The amount of fibroblast binding in each well after incubation for 1 hour was measured by the pNPP phosphatase assay. (e) The proliferation rate of shTEM1-transfected fibroblasts in 10% FBS was measured by WST-1 and BrdU assays. ***P < 0.001. P-values were determined by unpaired two-tailed Student t test, one-way or two-way ANOVA. ANOVA, analysis of variance. FBS, fetal bovine serum; pNPP, p-nitrophenyl phosphate; RT-PCR, reverse transcriptaseePCR; TEM1, tumor endothelium marker 1.

Article Snippet: Cell lysates (1,000 mg) were incubated with 1 mg anti-TEM1 antibody (18160-1-AP, Proteintech) or normal rabbit IgG (sc-2027, Santa Cruz Biotechnology) and 20 ml of protein G-agarose beads (MilliporeSigma, Temecula, CA) Journal of Investigative Dermatology (2019), Volume - at 4 C overnight.

Techniques: Migration, Knockdown, Transfection, shRNA, Expressing, Reverse Transcription Polymerase Chain Reaction, Wound Healing Assay, Transwell Migration Assay, Binding Assay, Incubation, Phosphatase Assay, Two Tailed Test, Marker

Journal: The Journal of investigative dermatology

Article Title: Tumor Endothelial Marker 1 (TEM1/Endosialin/CD248) Enhances Wound Healing by Interacting with Platelet-Derived Growth Factor Receptors.

doi: 10.1016/j.jid.2019.03.1149

Figure Lengend Snippet: Figure 5. The engagement of TEM1 in the downstream signaling pathway and mitogenic effects of PDGF on fibroblasts. (a, b) The amount of protein expression in shLuc- and shTEM1-transfected NIH3T3 cell lysates treated with different doses of PDGF-BB were analyzed using western blotting. A representative blot of p-ERK and p-Akt relative to ERK and Akt individually was shown with quantification by the Image J software. (c) Transwell migration assay was performed to observe the migration number in shLuc and shTEM1 NIH3T3 cells in the medium containing 0.5% FBS with or without administration of 50 ng/ml PDGF-BB in the lower chamber. Scale bar ¼ 100 mm. (d) WST-1 assay was used to study the proliferation of shLuc and shTEM1 NIH3T3 cells in medium containing 0.25% FBS with or without administration of 50 ng/ml PDGF-BB. *P < 0.05, **P < 0.01, and ***P < 0.001. P-values were determined by one-way or two-way ANOVA. Akt, protein kinase B; ANOVA, analysis of variance; ERK, extracellular signaleregulated kinase; FBS, fetal bovine serum; PDGF, platelet- derived growth factor; p-Akt, phosphorylated Akt; p-ERK, phosphorylated ERK; shLuc, luciferase short hairpin RNA; shTEM1, TEM1 short hairpin RNA; TEM1, tumor endothelium marker 1.

Article Snippet: Cell lysates (1,000 mg) were incubated with 1 mg anti-TEM1 antibody (18160-1-AP, Proteintech) or normal rabbit IgG (sc-2027, Santa Cruz Biotechnology) and 20 ml of protein G-agarose beads (MilliporeSigma, Temecula, CA) Journal of Investigative Dermatology (2019), Volume - at 4 C overnight.

Techniques: Expressing, Transfection, Western Blot, Software, Transwell Migration Assay, Migration, WST-1 Assay, Derivative Assay, Luciferase, shRNA, Marker

Journal: The Journal of investigative dermatology

Article Title: Tumor Endothelial Marker 1 (TEM1/Endosialin/CD248) Enhances Wound Healing by Interacting with Platelet-Derived Growth Factor Receptors.

doi: 10.1016/j.jid.2019.03.1149

Figure Lengend Snippet: Figure 6. Colocalization and coimmunoprecipitation of TEM1 and PDGFRa in NHDFs. (a) TEM1 (green) and PDGFRa (red) expression in the wound at 0 and 14 days in mice was detected with immunofluorescence staining. Scale bar ¼ 200 mm. (b) The expression of TEM1 and PDGFR relative to DAPI was quantified by the Image J software. (c) After NHDFs were supplemented with 20 ng/ml PDGF-BB for 0 or 15 minutes, TEM1 (green) and PDGFRa (red) expression was detected by confocal microscopy. The nucleus was stained with DAPI. Scale bar ¼ 10 or 50 mm. (d) Lysates from NHDFs in medium with 10% FBS were coimmunoprecipitated with the TEM1 antibody and immunoblotted with the PDGFRa antibody. (e) A proposed scheme depicting the biological function of TEM1 in the activated fibroblasts in the wound. *** and ### P < 0.001. P-values were determined by unpaired two-tailed Student t test. NHDF, normal human dermal fibroblast; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; TEM1, tumor endothelium marker 1.

Article Snippet: Cell lysates (1,000 mg) were incubated with 1 mg anti-TEM1 antibody (18160-1-AP, Proteintech) or normal rabbit IgG (sc-2027, Santa Cruz Biotechnology) and 20 ml of protein G-agarose beads (MilliporeSigma, Temecula, CA) Journal of Investigative Dermatology (2019), Volume - at 4 C overnight.

Techniques: Expressing, Staining, Software, Confocal Microscopy, Two Tailed Test, Derivative Assay, Marker

Journal: Scientific reports

Article Title: Musashi-1 Enhances Glioblastoma Cell Migration and Cytoskeletal Dynamics through Translational Inhibition of Tensin3.

doi: 10.1038/s41598-017-09504-7

Figure Lengend Snippet: Figure 3. Gene ontology (GO) and pathway analyses for MSI1 targets distribution in actin rearrangement. Ribonucleoprotein immunoprecipitation sequencing (RIP-seq) of MSI1-associated RNAs were performed in 05MG cells. (A) Gene ontology (GO term) enrichment analysis was conducted by DAVID software per the biological processed. (B) MSI1-immunoprecipitated mRNAs are enriched for protein-protein interaction network, of which the top-ranked genes from the GO terms were listed. (C) Non-significant genes were cut off, narrowing down the original 22667 genes into 2286 target genes. TNS3 was found in the highly expressed pool of genes. (D) The top 10 significantly changed genes were analyzed by REVIGO (http://revigo.irb.hr/) and listed in the chart. The numbers on each graph showed the corresponding GO term of cell migration and its read counts by FPKM (fragments per kilobase of exon per million fragments mapped). (E) Endogenous MSI1 was precipitated from 05MG cells treated without or with RNaseA (10 mg/ml). The precipitated complexes were subjected to RT-PCR to assess TNS3 mRNA levels and by Western blotting to confirm the MSI1 precipitation, of which the uncropped blots were demonstrated in Supplementary Figure 5.

Article Snippet: The complete cDNA sequence of TNS3 (NCBI accession no. NM_022748) was amplified and cloned into pCMV6-Entry expression vector (Cat# PS100001, OriGene Technologies, Rockville, MD, USA).

Techniques: Immunoprecipitation, Sequencing, Software, Migration, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Scientific reports

Article Title: Musashi-1 Enhances Glioblastoma Cell Migration and Cytoskeletal Dynamics through Translational Inhibition of Tensin3.

doi: 10.1038/s41598-017-09504-7

Figure Lengend Snippet: Figure 4. MSI1 repressed TNS3 translation through binding to its 3′UTR. GBM 05MG cells were transiently transfected with 0, 60 and 120 ng of siMSI1. TNS3 and MSI1 expression levels were analyzed 48 hours after transfection by (A) Western blotting and (B) Real-time PCR. U251 cells were transient transfected with 0, 0.5 and 1 μg Flag-MSI1. TNS3 and MSI1 expression levels were analyzed 48 hours after transfection by Western blotting (left), and the results were quantified as bar chart (right) and Real-time PCR (D). (E,F) RNA-ChIP assay was performed in endogenous MSI1 in 05MG cells and exogenous Flag-MSI1 in MSI1-overexpressed U251 cells. The bar chart indicates the gene expression fold change after normalization to IgG-precipitated controls. (G) Schematic representation of the reporter constructs containing the firefly luciferase fused to wild-type or mutated TNS3-3′UTR. (H) Luciferase reporter assays were conducted in U251 cells with or without MSI1 expression. The bioluminescent signal of each TNS3-3′UTR reporter were shown as relative fold change to their respective luciferase positive control. (I) Schematic illustration showing the sucrose gradient centrifugation. The gradient used in this study was 15% at the top and 45% at the bottom. Whole-cell lysates are

Article Snippet: The complete cDNA sequence of TNS3 (NCBI accession no. NM_022748) was amplified and cloned into pCMV6-Entry expression vector (Cat# PS100001, OriGene Technologies, Rockville, MD, USA).

Techniques: Binding Assay, Transfection, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Gene Expression, Construct, Luciferase, Positive Control, Gradient Centrifugation

Journal: Scientific reports

Article Title: Musashi-1 Enhances Glioblastoma Cell Migration and Cytoskeletal Dynamics through Translational Inhibition of Tensin3.

doi: 10.1038/s41598-017-09504-7

Figure Lengend Snippet: Figure 5. MSI1/TNS3-regulated GBM cell migration and actin remodeling is potentially governed by RhoA- GTP activation. (A) MSI1 and TNS3 protein levels were assessed in 05MG cells transiently transfected with siMSI1 (clone #1 and #2) and/or siTNS3 (clone #1 and #2) using Western blotting. (B) MSI1- and TNS3- depleted 05MG cells were subjected to TransWell migration assay. The number of migratory cells were counted and presented as percentages relative to the controls. The percentage of cell numbers were indicated by alamar blue assay shown in bar chart. (C) MSI1-mediated actin remodeling with TNS3 inhibition were shown with the immune-stained F-actin distribution in MSI1- and TNS3-depleted 05MG cells (left); the formation and the angular distribution of F-actin orientation were analyzed by assessing and quantifying the immunofluorescence intensities using Matlab software. Mean of Kurtosis was shown on the right (n = 45). (D) GTP-bound RhoA was pulled down (IP) with Rhotekin-RBD agarose beads from cell lysates and blotted with anti-RhoA antibodies. The intensities of RhoA-GTP were normalized to that of total RhoA and presented as relative fold change in the graph. (E) MSI1- and TNS3-depleted 05MG cells were subjected to Western blot for MSI1, TNS3 and RhoA protein levels. The intensity of RhoA-GTP blot at each group be calculating and presented as a relative ratio in comparison to the control group (siCON.). (F) TNS3-overexpressed (mycTNS3) and MSI1-overexpressed

Article Snippet: The complete cDNA sequence of TNS3 (NCBI accession no. NM_022748) was amplified and cloned into pCMV6-Entry expression vector (Cat# PS100001, OriGene Technologies, Rockville, MD, USA).

Techniques: Migration, Activation Assay, Transfection, Western Blot, Transwell Migration Assay, Alamar Blue Assay, Inhibition, Staining, Immunofluorescence, Software, Comparison, Control

Journal: Scientific reports

Article Title: Musashi-1 Enhances Glioblastoma Cell Migration and Cytoskeletal Dynamics through Translational Inhibition of Tensin3.

doi: 10.1038/s41598-017-09504-7

Figure Lengend Snippet: Figure 6. MSI1/TNS3 signaling promoted in vivo tumor migration and correlated with poor patient survival. (A,B) In vivo GBM growth rate was assessed by subcutaneous tumor implantation in immunocompromised mice with 5 × 105 MSI1-depleted 05MG cells or 5 × 105 MSI1-overexpressed U251 cells, each facilitated with their respective controls. Tumor volume was measured by caliper. Data were presented as mean ± SEM (n = 12). *p < 0.05 (relative to the control group). (C) SCID mice were orthotopically implanted with 1.5 × 105 GFP- labeled U251-Flag-control or 1.5 × 105 GFP-labeled U251-MSI1-ovexpressed cells. Representative photographs of fluorescent images were taken 63 days after inoculation. The GFP-labeled GBM tumors can be observed and the tumor sections were subjected to H&E, IHC, and IF staining to assess tumor malignancy, spreading, as well as the expression of MSI1 and TNS3. (D) Tumor tissue was harvested and homogenized for western blotting to assess MSI1 and TNS3 protein levels ex vivo. The uncropped blots were demonstrated in Supplementary Figure 5. Correlation of survival rates and MSI1/TNS3 gene expression in GBM patients. (E) Heat map of MSI1 or TNS3 mRNA expression of GBM patient samples along with the risk factor. The high and low risk groups

Article Snippet: The complete cDNA sequence of TNS3 (NCBI accession no. NM_022748) was amplified and cloned into pCMV6-Entry expression vector (Cat# PS100001, OriGene Technologies, Rockville, MD, USA).

Techniques: In Vivo, Migration, Tumor Implantation, Control, Labeling, Staining, Expressing, Western Blot, Ex Vivo, Gene Expression