Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Distribution of EGF and EGFR and phosphorylation status of EGFR in EGF treated cells. ( A ) ICC for EGF and EGFR in untreated cells and cells treated with EGF for 30 min Confocal imaging was done on laser scan confocal Nikon Ti microscope at 100x magnification; staining was done for nuclei (Hoechst 33342), EGF (ab9595 Abcam, Cambridge, United Kingdom) and EGFR (antibody recognizing extracellular domain of human EGFR: epitope 6-273 aa, antibody clone sc-101; Santa Cruz Biotechnology, TX, United States). The scale bars are 50 μm. ( B ) Phosphorylation of EGFR upon treatment with EGF was determined by Western blot. Whole cell lysates were obtained from cells without treatment or 30 min after treatment with recombinant EGF. Three independent experiments were done and imaged using Li-Cor system; representative Western blots are shown. Normalization was done using α-Tubulin as a loading control (Catalog number 926-42213 Li-Cor, NE, United States). Antibodies for phosphorylated EGFR included Y1068 (Catalog number #3777, Cell Signaling, MA, United States), Y1086 (Catalog number #2220S, Cell Signaling, MA, United States) and Y1173 (Catalog number ab32578 Abcam, Cambridge, United Kingdom). ( C ) Western blot quantification was done using software provided by Li-Cor and plotted using Graph Pad Prizm. This bar graph represents relative quantification of phosphorylation of EGFR (for tyrosine positions Y1068, Y1086 and Y1173) for wild type ME180 cells and mutant clone VII11 at 30 min after EGF treatment. To show relative phosphorylation values, the highest phosphorylation signal (that for Tyrosine 1068 in wild type cells) was set as 1. Results are presented as the means ± SD of at least three independent experiments.

Article Snippet: For the EGF and TGF-α binding test, cells were grown on glass cover slips and incubated with 1% BSA in McCoy’s serum free medium for 1 h, and then placed on ice for 10 min. After removal of the medium, either EGF AlexaFluor ® 555 (Invitrogen, MA, United States200 fold dilution of 1 μg/μl stock solution) or recombinant EGF protein (Catalog number 236-EG, R&D systems, MN, United States, stock 0.5 mg/ml, diluted 1:200), were diluted in McCoy’s Medium with 1% BSA and incubated with cells.

Techniques: Phospho-proteomics, Imaging, Microscopy, Staining, Western Blot, Recombinant, Control, Software, Quantitative Proteomics, Mutagenesis

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Evaluation of EGFR downstream signaling in wild type cells and clones upon treatment with EGF. ( A ) Western blots were performed on whole cell lysates isolated from cells serum starved for 1 h and then incubated with recombinant EGF for 30 min in serum free media with 1% BSA. Control cells were incubated in serum free media for 30 min. Experiments were performed independently from at least 3 different passages of cells and representative blots are shown. Antibodies used in this work came from antibody panel ab283852 (Abcam, Cambridge, United Kingdom). ( B ) Quantification of Western blot results was performed using Li-cor quantification software. The results were normalized to protein expression in wild type cells not treated with EGF set at 100%. Data are presented as the mean ± SD of at least three independent experiments.

Article Snippet: For the EGF and TGF-α binding test, cells were grown on glass cover slips and incubated with 1% BSA in McCoy’s serum free medium for 1 h, and then placed on ice for 10 min. After removal of the medium, either EGF AlexaFluor ® 555 (Invitrogen, MA, United States200 fold dilution of 1 μg/μl stock solution) or recombinant EGF protein (Catalog number 236-EG, R&D systems, MN, United States, stock 0.5 mg/ml, diluted 1:200), were diluted in McCoy’s Medium with 1% BSA and incubated with cells.

Techniques: Clone Assay, Western Blot, Isolation, Incubation, Recombinant, Control, Software, Expressing

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Evaluation of cell cycle distribution and expression of cell cycle regulator proteins. ( A ) Representative cell cycle profiles of wild type ME180 cells and mutant clones based on DAPI staining and incorporation of EdU. The usual horseshoe pattern cell distribution allowed separation of cell populations into G1, S and G2/M stage of the cell cycle. ( B ) Histogram showing cell cycle distribution in wild type cells and mutant clones based on flow cytometry data done on 5 independent experiments using cells from 5 different cell passages. The data are shown as means ± SD from five independent experiments including cells from three different cell cycle passages. ( C ) Western blots performed on whole cell lysates from wild type cells and mutant clones growing in cell culture or additionally treated with recombinant EGF for 30 min. Experiments were performed independently using at least three different cell passages and representative blots are shown. ( D ) Quantification of Western blot results was performed using Li-Cor quantification software and the results are presented values relative to protein expression in untreated wild type cells set as 100%. Data are presented as means ± SD of at least three independent experiments.

Article Snippet: For the EGF and TGF-α binding test, cells were grown on glass cover slips and incubated with 1% BSA in McCoy’s serum free medium for 1 h, and then placed on ice for 10 min. After removal of the medium, either EGF AlexaFluor ® 555 (Invitrogen, MA, United States200 fold dilution of 1 μg/μl stock solution) or recombinant EGF protein (Catalog number 236-EG, R&D systems, MN, United States, stock 0.5 mg/ml, diluted 1:200), were diluted in McCoy’s Medium with 1% BSA and incubated with cells.

Techniques: Expressing, Mutagenesis, Clone Assay, Staining, Flow Cytometry, Western Blot, Cell Culture, Recombinant, Software

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:

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