Journal: bioRxiv

Article Title: Histologic variants in bladder cancer harbor aggressive molecular features including TM4SF1 expression and a CA125+ cell state

doi: 10.1101/2024.05.20.595039

Figure Lengend Snippet: (A) Volcano plot comparison of all UC and HV cells after downsampling (N = 150 per patient). (B) Violin plots of TM4SF1 expression by tumor cluster. (C) Correlation plots of TM4SF1 and EMP1, EZR, CLDN4, and KRT19. (D-E) Immunohistochemistry of TM4SF1 in a validation cohort of HV and UC (D) primary tumors and (E) lymph node metastases. (F) Semiquantitative comparison of TM4SF1 staining in HV and UCs.

Article Snippet: Flow cytometric quantification of TM4SF1 expression across human bladder cancer cell lines was performed by incubating with anti-TM4SF1-PE antibody (Miltenyi, clone REA851, 1:100) for 30-60 minutes on ice.

Techniques: Comparison, Expressing, Immunohistochemistry, Biomarker Discovery, Staining

Journal: bioRxiv

Article Title: Histologic variants in bladder cancer harbor aggressive molecular features including TM4SF1 expression and a CA125+ cell state

doi: 10.1101/2024.05.20.595039

Figure Lengend Snippet: (A) Schematic for generating TM4SF1-CAR T cells. (B) Bladder cancer cell lines and TM4SF1 expression determined by flow cytometric fluorescent antibody detection and mRNA expression. (C) Quantification of in vitro TM4SF1-CAR1 and CAR2 activity against bladder cancer cell lines using IncuCyte co-culture assay with 1:1 effector:target cell ratio. (D) Schematic for xenograft generation from the UMUC3 cell line and in vivo TM4SF1-CAR1 testing. (E) Tumor size comparisons between TM4SF1-CAR treated and untreated mice. (F) Kaplan-Meier survival analysis of treated and untreated mice.

Article Snippet: Flow cytometric quantification of TM4SF1 expression across human bladder cancer cell lines was performed by incubating with anti-TM4SF1-PE antibody (Miltenyi, clone REA851, 1:100) for 30-60 minutes on ice.

Techniques: Expressing, In Vitro, Activity Assay, Co-culture Assay, In Vivo

Journal: Journal of Gastrointestinal Oncology

Article Title: Analyzing the role of TM4SF1 expression in pancreatic adenocarcinoma: understanding prognostic implications and therapeutic opportunities

doi: 10.21037/jgo-24-564

Figure Lengend Snippet: Differential gene expression and functional enrichment analysis of PAAD. (A) Volcano plot highlighting DEGs between PAAD and normal tissues. The most significant upregulated gene in the tumor tissues was TM4SF1 . Red dots signify genes that are highly expressed in the tumor group. Blue dots indicate genes with low expression in the tumor group. Gray dots, labeled as ‘not’, represent genes deemed non-differentially expressed due to not meeting the screening threshold. (B) Bubble plot depicting the GO and KEGG functional enrichment of the up- and downregulated DEGs. (C) GSEA analysis results illustrating the signaling pathways significantly enriched with DEGs. BP, biological process; CC, cellular component; MF, molecular function; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, normalized enrichment score; PAAD, pancreatic adenocarcinoma; DEGs, differentially expressed genes.

Article Snippet: Primary antibodies against β-actin (#AF0003, Beyotime) and TM4SF1 (#NBP1-76549, NOVUS, Colorado, USA) were incubated on the membranes overnight at 4 °C.

Techniques: Gene Expression, Functional Assay, Expressing, Labeling, Protein-Protein interactions

Journal: Journal of Gastrointestinal Oncology

Article Title: Analyzing the role of TM4SF1 expression in pancreatic adenocarcinoma: understanding prognostic implications and therapeutic opportunities

doi: 10.21037/jgo-24-564

Figure Lengend Snippet: Immune cell infiltration analysis between high- and low-TM4SF1 expression groups. (A) Scatter plot correlating TM4SF1 expression with levels of immune cell infiltration. (B) Correlation analysis reinforcing the findings in (A). (C) Differential immune cell infiltration based on ssGSEA analysis between the high- and low-TM4SF1 expression groups. (D) High- and low-risk immune features. *, P<0.05; **, P<0.01; ***, P<0.001. MDSC, myeloid-derived suppressor cell; ssGSEA, single-sample gene set enrichment analysis; L, low; H, high.

Article Snippet: Primary antibodies against β-actin (#AF0003, Beyotime) and TM4SF1 (#NBP1-76549, NOVUS, Colorado, USA) were incubated on the membranes overnight at 4 °C.

Techniques: Expressing, Derivative Assay

Journal: Journal of Gastrointestinal Oncology

Article Title: Analyzing the role of TM4SF1 expression in pancreatic adenocarcinoma: understanding prognostic implications and therapeutic opportunities

doi: 10.21037/jgo-24-564

Figure Lengend Snippet: Gene mutation analysis of the five key prognostic genes. (A) Chromosomal positions of the five key genes. (B) Forest plot illustrating the hazard ratios of the five genes concerning PAAD prognosis. (C) Correlation heatmap of the five key genes. (D) Distribution of the mutation frequencies across patients. (E) Waterfall plot of the genes ranked by mutation count. (F) Tissue samples from 10 patients were obtained to examine the mRNA expression differences of TM4SF1, BPIFB4, CPTP, DVL1, PLEKHN1 , and DDR1 between cancer and normal tissues. **, P<0.01. SNP, single nucleotide polymorphism; INS, insertion; DEL, deletion; SNV, single-nucleotide variant; PAAD, pancreatic adenocarcinoma; mRNA, messenger RNA; HR, hazard ratio.

Article Snippet: Primary antibodies against β-actin (#AF0003, Beyotime) and TM4SF1 (#NBP1-76549, NOVUS, Colorado, USA) were incubated on the membranes overnight at 4 °C.

Techniques: Mutagenesis, Expressing, Variant Assay

Journal: Journal of Gastrointestinal Oncology

Article Title: Analyzing the role of TM4SF1 expression in pancreatic adenocarcinoma: understanding prognostic implications and therapeutic opportunities

doi: 10.21037/jgo-24-564

Figure Lengend Snippet: TM4SF1 expression and its functional effects on pancreatic cancer cell behavior. (A) Box plot depicting the relative expression of TM4SF1 mRNA in pancreatic tumor tissues compared to normal tissues, demonstrating a significant upregulation in tumors (*, P<0.05). (B) Kaplan-Meier survival curves stratifying patients into quartiles based on TM4SF1 expression levels, showing an inverse relationship between high TM4SF1 expression and overall survival. (C) Western blot analysis of TM4SF1 protein levels in normal (N1–N5) and tumor (T1–T5) pancreatic tissue samples, with tumors exhibiting higher expression levels. (D) Western blot comparison of the TM4SF1 protein across a panel of pancreatic cancer cell lines, identifying varying expression levels. (E) Bar graph quantifying TM4SF1 mRNA expression in various pancreatic cancer cell lines, with PANC-1 cells showing the highest expression. (F,G) Bar graph showing TM4SF1 mRNA levels in PANC-1 cells after treatment with NC or TM4SF1 -targeting siRNAs (si TM4SF1 #1, #2 and #3), indicating effective knockdown. Proliferation assay (H) and colony formation assay (1×) (stained with 0.1% crystal violet) (I) of PANC-1 cells following si TM4SF1 treatment, revealing reduced cell growth and colony number compared to NC. (J) Cell viability assay assessing the sensitivity of PANC-1 cells to Gemcitabine post-si TM4SF1 treatment, showing increased drug sensitivity. Transwell migration, invasion (10×) (stained with 0.1% crystal violet) (K) and cellular wound healing (5×) (L) assays in si TM4SF1 -treated PANC-1 cells demonstrated decreased migratory and invasive capacities. (M) Western blot confirming overexpression of TM4SF1 in CFPAC-1 cells transfected with oe TM4SF1 versus NC. Cell proliferation assay (N) and colony formation assay (1×) (stained with 0.1% crystal violet) (O) in CFPAC-1 cells with oe TM4SF1 showed enhanced growth and colony-forming ability. Transwell migration, invasion (10×) (stained with 0.1% crystal violet) (P) and cellular wound healing (5×) (Q) assays in oe TM4SF1 -expressing CFPAC-1 cells indicated increased migration and invasion potential. *, P<0.05; **, P<0.01; ***, P<0.001. TPM, transcripts per million; PAAD, pancreatic adenocarcinoma; HR, hazard ratio; NC, control; IC 50 , half-maximal inhibitory concentration; oeTM4SF1, overexpression of TM4SF1; mRNA, messenger RNA.

Article Snippet: Primary antibodies against β-actin (#AF0003, Beyotime) and TM4SF1 (#NBP1-76549, NOVUS, Colorado, USA) were incubated on the membranes overnight at 4 °C.

Techniques: Expressing, Functional Assay, Western Blot, Comparison, Knockdown, Proliferation Assay, Colony Assay, Staining, Viability Assay, Migration, Over Expression, Transfection, Control, Concentration Assay

Journal: Developmental biology

Article Title: Deletion of the Dishevelled family of genes disrupts anterior-posterior axis specification and selectively prevents mesoderm differentiation.

doi: 10.1016/j.ydbio.2020.05.010

Figure Lengend Snippet: Fig. 6. Effects of Inhibiting BMP, Canonical Wnt, and Nodal signaling on Germ Lineage Differentiation. A-B) Representative images of Brachyury (green), Otx2 (red), and DAPI (blue) immunostaining of WT and Dvl TKO EBs on day 7 of differentiation after inhibition with BMP (LDN-193189), canonical and non-canonical Wnt (IWP L6) and Nodal (SB-431542) drug treatments. Scale bar ¼ 100 μm. C-D) Quantification of Brachyury and Otx2-positive cells in WT and Dvl EBs differentiated for 5–7 days (n ¼ 20/group). Brachyury-positive cells are not detectable in Dvl TKO EBs, and there are no significant differences in Otx2-positive cells. E-F) Quantitative RT-PCR analysis of Brachyury (mesoderm) and GATA4 (endoderm) mRNA expression to determine differences in differentiation potential in WT and Dvl TKO EBs over a 6-day time course (n ¼ 3). Expression is normalized to the untreated controls in Fig. 5G (Brachyury) and 5H (GATA4). E) Brachyury expression in WT EBs is attenuated with inhibition of BMP, Wnt, and Nodal signaling, similar to Dvl TKO EBs. F) Inhibition of BMP and Wnt signaling significantly enhances endoderm differentiation in WT and Dvl TKO EBs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The following drug treatments were added at the time of plating and removed after 48 h: 100 ng/ml Activin A (R&D Systems), 50 ng/ml BMP4 (R&D Systems), 3uM CHIR-99021 (Tocris), 100 ng/ml Wnt3a (R&D Systems), 10 μM SB-431542 (EMD Millipore), 200 nM LDN-193189 (Stemgent), 0.5 μM IWP L6 (Tocris).

Techniques: Immunostaining, Inhibition, Quantitative RT-PCR, Expressing

Journal: Developmental biology

Article Title: Deletion of the Dishevelled family of genes disrupts anterior-posterior axis specification and selectively prevents mesoderm differentiation.

doi: 10.1016/j.ydbio.2020.05.010

Figure Lengend Snippet: Fig. 7. Effects of Developmental Pathway Modulation on Germ Lineage Differentiation. Activation of BMP (BMP4), canonical Wnt (Wnt3a and CHIR-99021) and Nodal (Activin A) pathways in WT EBs enhances mesoderm (Bra) differentiation, while endoderm (GATA4) differentiation remains low, and ectoderm (Otx2) dif- ferentiation diminishes over time. Treatment with these activators in Dvl TKO EBs does not rescue mesoderm induction. The relative level of endoderm marker expression is higher in Dvl TKO EBs compared to WT EBs treated with Wnt activators, Wnt3a and CHIR, but not BMP4 or Activin A. Dvl loss also delays the decrease in ectoderm lineage expression. Inhibition of BMP (LDN-198189), canonical and non-canonical Wnt (IWP L6) and Nodal (SB431542) pathways in WT EBs results in the suppression of mesoderm differentiation, recapitulating the Dvl TKO phenotype. Pathway inhibitor treatment in WT EBs, leads to similar trends of differentiation as Dvl TKO EBs. Endoderm differentiation is greatly enhanced in Dvl TKO EBs with LDN-193189 and IWP L6 treatment.

Article Snippet: The following drug treatments were added at the time of plating and removed after 48 h: 100 ng/ml Activin A (R&D Systems), 50 ng/ml BMP4 (R&D Systems), 3uM CHIR-99021 (Tocris), 100 ng/ml Wnt3a (R&D Systems), 10 μM SB-431542 (EMD Millipore), 200 nM LDN-193189 (Stemgent), 0.5 μM IWP L6 (Tocris).

Techniques: Activation Assay, Marker, Expressing, Inhibition

Journal: The European Respiratory Journal

Article Title: Airway-derived emphysema-specific alveolar type II cells exhibit impaired regenerative potential in COPD

doi: 10.1183/13993003.02071-2023

Figure Lengend Snippet: Decreased progenitor cell function of emphysema-specific alveolar type II (ATII) cells in human COPD lungs. a) Detection of asATII cells in alveolar sacs of emphysematous region (black square in left picture with haematoxylin and eosin (H&E) staining) by co-immunofluorescence (co-IF) for CD74 (green), secretaglobin-3A2 (SCGB3A2) (red), and surfactant protein C (SPC) (white). Scale bars=200 µm, 50 µm and 10 µm. b) Detection of rbATII cells in respiratory bronchiole of emphysematous region (black square in left picture with H&E staining) by co-IF for intercellular adhesion molecule 1 (ICAM1) (green, top), TM4SF1 (green, bottom), SCGB3A2 (red), and SPC (white). Scale bars=50 µm, 20 µm and 10 µm. c) Flow cytometry analysis of asATII and rbATII using CD74 (centre), TM4SF1 and ICAM1 (right) in healthy control sample (n=6, top) and emphysematous parenchymal tissue from COPD patients (n=6, bottom). d) Quantification of the percentages of asATII (CD74 + ) and rbATII (ICAM1 + /TM4SF1 + ) cells in total ATII cells (HTII280 + /epithelial cell adhesion molecule (EPCAM) + /4′,6-diamidino-2-phenylindole (DAPI) − ) in a). Mann–Whitney test. e) Organoids formed by fluorescence-activated cell sorted asATII (HTII280 + /CD74 + ) and rbATII (HTII280 + /ICAM1 + /TM4SF1) cells from human emphysematous parenchymal tissue (n=5) and by ATII cells (HTII280 + ) from healthy control (n=3). Scale bar=100 µm. f) Quantification of organoid forming efficiency of c). Values are presented as mean± sem . Mann–Whitney test. *: p<0.05.

Article Snippet: Primary antibodies used for immunofluorescence include rabbit anti-SPC (Millipore, AB3786), rabbit anti-SPC (Abcam, ab90716), mouse IgM anti-HTII280 (Terrace Biotech, TB-27AHT2-280), CD74 (Novus, NBP1-33109), intercellular adhesion molecule 1 (ICAM1) (Novus, BBA3), TM4SF1 (R&D Systems, MAB8164), SCGB3A2 (R&D Systems, AF3545), acetylated α-tubulin (Abcam, ab24610), tdTomato (Biorbyt, orb182397) and chicken anti-Ly6i antibody.

Techniques: Cell Function Assay, Staining, Immunofluorescence, Flow Cytometry, Control, MANN-WHITNEY, Fluorescence

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: Real-time PCR primer sequences used in the study

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: Locally applied EGFL6 accelerates bone formation and consolidation in a rat model of tibia distraction osteogenesis (DO). a Overall schematic diagram illustrating the study design. DO was performed in three phases as indicated. Midway through the distraction phase on day 10, recombinant EGFL6 (200 ng/ml), or an equivalent volume of sterile PBS (control), was infused into the distracted area and then infused again every 2 days until the end of the distraction phase on day 15. Distraction was performed at a rate of 0.25 mm per 12 h. Asterisk (*) in a indicates that the tibia bone fragments were distracted for a total of 5 mm over a period of 10 days. b X-ray images (lateral view) of the distracted bones from representative cases after 2, 3, and 4 weeks of consolidation. Bright white angular areas in images are the densities of the metal monolateral external fixator. c, d Three-dimensional reconstructions ( c ) and internal longitudinal profiles ( d ) derived from micro-CT of distracted tibia bones from representative cases of EGFL6-treated and control rats after 2 and 4 weeks of consolidation. Light areas show the increased bone-tissue mineralization. e, f Quantitation analysis of bone-tissue mineralization showing the mean (±SD) percentage bone volume/total tissue volume (BV/TV) and mean (±SD) bone mineral density (BMD) in EGFL6-treated and control rats. Mineralization parameters were calculated from the micro-CT image data. Significant differences were evaluated by one-way ANOVA with post hoc Dunnett’s tests. * p < 0.05

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Recombinant, Sterility, Control, Derivative Assay, Micro-CT, Quantitation Assay

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: Effects of different concentrations of EGFL6 protein on human umbilical cord vein endothelial cell (HUVEC) angiogenesis in vitro. a Phase-contrast images of HUVEC cultures treated with EGFL6 showing cell migration in the scratch-wound assay at the indicated times. Vertical dashed lines (white) demarcate the border between the wavefront of migrating cells and scratched area that was initially void of cells. b Quantitation (mean ± SD) of cell proliferation in response to EGFL6 (CCK-8 assay). c Mean percentage of cells migrating as a function of EGFL6 concentration in the scratch-wound assay. d, e Crystal violet-stained HUVECs that migrated in the transwell assay. Optical density (OD) of staining is relative to untreated control cells ( e ). f Relative quantification of capillary-like structures formed by HUVECs cultured with EGFL6 in the tube-formation assay. Values are relative to control values. g Phase-contrast images of HUVECs cultured with EGFL6 in the tube-formation assay. h Expression levels of Hif1a, VEGF-A, CD31, and EMCN genes in HUVECs treated with EGFL6 for 1 day, as evaluated by RT-PCR. The housekeeping gene GAPDH served as an internal control. i, j Quantitation of VEGF-A protein concentration in HUVECs treated with EGFL6 (200 ng/ml) for the indicated times. k, l Western blots of lysates from HUVECs treated with EGFL6. Blots were probed with antibodies against angiogenesis markers (Hif1a, VEGF-A, CD31, EMCN) and pathway markers (β-catenin, pβ-catenin, active β-catenin, and pGSK3β). GADPH is the loading control. Significant differences among groups were determined by one-way ANOVA and post hoc Dunnett’s test; * p < 0.05; ** p < 0.01; and *** p < 0.001. All immunoblots were cropped from the original here and in subsequent figures. Experimental HUVECs were treated with the indicated EGFL6 concentrations. Control and experimental conditions for all functional assays were the same, except controls lacked EGFL6. Histogram values are based on three replicated experiments, and error bars are SD here and in all subsequent figures. Scale bars for a, e, g , 250 μm

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: In Vitro, Migration, Scratch Wound Assay Assay, Quantitation Assay, CCK-8 Assay, Concentration Assay, Staining, Transwell Assay, Control, Quantitative Proteomics, Cell Culture, Tube Formation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Protein Concentration, Western Blot, Functional Assay

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: EGFL6 treatment enhances osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. a BMSC viability after treatment with different concentrations of EGFL6, as assessed by the CCK-8 assay. Values are means±SD. b Images of Alizarin Red S (AR-S)-stained BMSCs treated with EGFL6, showing increased mineralization (rust-colored deposits). Osteogenic differentiation of BMSCs was examined on day 14. c Images of alkaline phosphatase (ALP)-stained BMSCs treated with EGFL6. Osteogenic differentiation of BMSCs was examined on day 3. Insets in b and c show low-magnification images of entire culture well. Scale bars, 250 μm. BMSCs were treated with different concentrations of EGFL6 for 5 or 10 days. d Expression levels of angiogenesis- and osteogenesis-related markers in BMSCs following treatment with/without EGFL6 for 5 days, as evaluated by RT-PCR. The housekeeping gene GAPDH served as an internal control. e Western blots of lysates from cultured BMSCs treated with/without EGFL6 for 5 or 10 days. Blots were probed with antibodies against different markers for angiogenesis (VEGF-A), osteogenesis (BMP2, CXCR4, RUNX2), and the Wnt/b-catenin signaling pathway (b-catenin, pb-catenin, active β-catenin, and pGSK3β). f Quantitation of expression of angiogenesis-, osteogenesis-, and pathway-related marker proteins in panel e . Significant differences among groups were determined by one-way ANOVA and post hoc Dunnett’s test; * p < 0.05; ** p < 0.01; and *** p < 0.001

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: In Vitro, CCK-8 Assay, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Cell Culture, Quantitation Assay, Marker

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: EGFL6 treatment enhances osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro . a, c, e Immunofluorescent images of EGFL6-treated BMSCs stained for the osteogenic-specific protein RUNX2 ( a ), and pathway-specific protein β-catenin ( c ) and active β-catenin ( e ). Cells were counterstained with the nuclear stain DAPI (blue) and the cytoskeleton stain phalloidin (red). Scale bars, 100 μm. b, d, f Quantitation of mean relative levels of RUNX2 ( b ), β-catenin ( d ), and active β-catenin ( f ) in BMSCs treated with EGFL6 (200 ng/ml). Significant differences between experimental and control groups were evaluated by Student t tests; * p < 0.05; ** p < 0.01; and *** p < 0.001

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: In Vitro, Staining, Quantitation Assay, Control

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: Dickkopf-related protein 1 (DKK1) partially suppresses EGFL6-enhanced BMSC osteogenesis in vitro. a Light micrographs of ALP-stained BMSC cultures on day 3 of differentiation. BMSCs were treated with 200 ng/ml EGFL6 to enhance osteogenic differentiation, and then supplemented with/without 0.3 μg/ml DKK1. Insets show low-magnification images of entire culture well. Scale bars, 250 μm. b AR-S staining of differentiated BMSCs showing mineralization (red) on day 14 after DKK1 application. Insets show low-magnification images of entire culture well. Scale bars, 250 μm. c Western blots showing the expression of osteogenic-specific and Wnt/β-catenin signaling-related proteins in BMSCs treated with/without EGFL6 and with/without DKK1. GADPH is the loading control. d Quantitation of osteogenic-specific and Wnt/β-catenin signaling-related proteins normalized to control condition (NS; black-colored bars). e, f, g Immunofluorescent images of BMSCs stained for RUNX2 (green) or active β-catenin (green). BMSCs were cultured with 200 ng/ml EGFL6 to enhance BMSC osteogenesis, and then treated with/without 0.3 μg/ml DKK1, an antagonist of Wnt/β-catenin signaling. Scale bar, 100 μm. Quantitation of RUNX2 or active β-catenin immunofluorescent staining showing mean relative fluorescence of DKK1 + EGFL6 (blue-colored bars) and EGFL6 alone (pink-colored bars) conditions normalized to control fluorescence (no DKK1, no EGFL6; gray-colored bars). Significant differences were evaluated by one-way ANOVA and post hoc Dunnett’s tests for all panels; * p < 0.05; ** p < 0.01; and *** p < 0.001

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: In Vitro, Staining, Western Blot, Expressing, Control, Quantitation Assay, Cell Culture, Fluorescence

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: EGFL6 stimulated formation of bone after consolidation for 2 and 4 weeks in a rat tibia DO model. a–c Images of histological sections of regenerated bone obtained from the distraction zone (boxed areas in a ) of rats treated with EGFL6 or PBS (control). Sections were stained with hematoxylin and eosin (HE) ( a ), Masson trichrome stain ( b ), or Safranin O/Fast green stain ( c ) in order to indicate newly formed trabecular bone, fibrous tissue, and cartilaginous tissue, respectively, in the distracted area. Scale bars, 200 μm

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Control, Staining

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: EGFL6 stimulated formation of bone and CD31 hi EMCN hi -positive type H vessels after consolidation for 2 and 4 weeks in a rat tibia DO model. a Sections were immunostained for osteocalcin (OCN), a hormone secreted by osteoblasts, and visualized with peroxidase-DAB. Quantitation of OCN-positive staining intensity in the distraction zone is summarized in the histograms on the right. b Sections were immunostained for VEGF-A, a key angiogenesis marker, and visualized with peroxidase-DAB. Quantitation of VEGF-A-positive staining intensity in the distraction zone after consolidation for 2 weeks is summarized in the histograms on the right. c Immunofluorescent images of regenerated bone sections obtained from the distraction zone immunostained for active β-catenin (green). The sections were counterstained with DAPI (blue), which stains nuclei of all cells. d Immunofluorescent images of regenerated bone sections obtained from the distraction zone immunostained for CD31 (red) or endomucin (EMCN, green). The sections were counterstained with DAPI (blue). Note that CD31 hi EMCN hi (yellow) vessels in EGFL6-treated rats are densely stained (arrows) compared to vessels in the controls. Scale bars for a–d , 200 μm. Significant differences were evaluated by Student t tests; * p < 0.05

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Quantitation Assay, Staining, Marker

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: Working model of EGFL6-mediated signaling, illustrating the coupling of angiogenesis and osteogenesis in the rat DO model. During the consolidation phase of bone remodeling, type H vessels form alongside newly developing bone and extend toward the distraction gap. In the bone marrow microenvironment, multiple cell types secrete angiogenic factors to support type H vessel formation. Osteoblast-lineage cells and ECs secrete VEGF-A. EGFL6 secreted by osteoblasts enhances VEGF-A expression in ECs to promote cell migration, tube formation, and branching, which further stimulates the formation of type H vessels during early consolidation phase. As a key regulatory factor, EGFL6 also promotes osteogenic differentiation of BMSCs into osteoblast-lineage cells, activated by the Wnt/β-catenin signaling pathway. EGFL6 also increases expression of the osteogenic proteins RUNX2, BMP2, and OCN, leading to faster restoration of the bone defect in the DO model. Abbreviations: ECs, endothelial cells; BMSCs, bone marrow mesenchymal stem cells; EGFL6, epidermal growth factor-like domain-containing protein 6; VEGF-A, vascular endothelial growth factor; RUNX2, Runt-related transcription factor 2; BMP2, bone morphogenetic protein 2; OCN, osteocalcin

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Expressing, Migration

Journal: Materials Today Bio

Article Title: Injectable hyaluronate-L- cysteine gel potentiates photothermal therapy in osteosarcoma via vorinostat-copper cell death

doi: 10.1016/j.mtbio.2024.101368

Figure Lengend Snippet: An Illustrative Depiction of the Fabrication of CHASA and Its Promising Anti-Tumor Mechanism, Featuring Cuproptosis Potentiated by Copper-Death Enhancer SAHA and Photothermal Effect Triggered by 808 nm near-infrared laser. Abbreviations: poly-Cys (hyaluronate-L-cysteine hydrogel precursors, poly-HA-Cys), SAHA (Vorinostat), CHASA (SAHAm@{[Cu(HA-Cys) 2 ]Cl 2 }n hydrogel), SLC31A1(Solute Carrier Family 31 Member 1), ATP7A/B(Copper-transporting P-type ATPase)，TCA cycle (Tricarboxylic Acid Cycle), DLAT (Dihydrolipoamide S-Acetyltransferase)，LIAS(Lipoic Acid Synthase)，LA (Lipoic Acid).

Article Snippet: Vorinostat (SAHA, Cat no.8638-EG-050) was purchased from R&D Systems Inc., Minneapolis, MN, USA.

Techniques: