Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A Workflow for comprehensive identification of transcriptionally active lncRNAs in the gastric mucosa of patients with GC. B Pie chart depicting gene loci harboring H3K4me3 peaks at transcription start sites (TSSs). These loci were categorized as protein-coding genes, lncRNA genes, or other genes. Peaks were categorized as patient-specific, healthy individual-specific, and common. C H3K4me3 peaks of representative genes in each category. D Summarized qRT-PCR results for 15 selected lncRNAs from eight GC cell lines. E qRT-PCR analysis of TCONS_00006264 (TM4SF1-AS1) and TM4SF1 in GC cell lines and normal stomach tissue. The locations of these two genes are shown at the top. ( n = 3). F Expression of TM4SF1-AS1 in primary GCs ( n = 374) and normal stomach tissues ( n = 32) from The Cancer Genome Atlas (TCGA) dataset. * P < 0.05.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Quantitative RT-PCR, Expressing

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A qRT-PCR analysis of TM4SF1-AS1 in the indicated GC cell lines transfected with siRNAs targeting TM4SF1-AS1 (si-1 and 2) or a control siRNA (si-Ctrl). ( n = 3). B Results of cell viability assays with GC cell lines transfected with the indicated siRNAs. ( n = 8). C qRT-PCR analysis of TM4SF1-AS1 in HSC-45 cells with inducible shRNAs targeting TM4SF1-AS1 (sh-1 and 2) or a control shRNA (sh-Ctrl). Cells were incubated for 8 days with or without doxycycline (Dox). ( n = 3). D Colony formation assays using HSC-45 cells with inducible shRNAs. Cells were incubated for 8 days with or without Dox. Summarized results are shown on the right; error bars represent SDs. ( n = 3). E Tumor growth in mice injected with HSC-45 cells inducibly expressing the indicated shRNAs. Mice were treated with or without Dox. Growth curves (left), resected tumors (middle), and tumor weights (right) are shown. F qRT-PCR analysis of TM4SF1-AS1 in the resected tumors in E . ( n = 4). G Tumor growth in mice injected with SNU638 cells stably expressing GFP or TM4SF1-AS1. Growth curves are shown on the left, and resected tumors are shown on the right. ( n = 4). H qRT-PCR analysis of TM4SF1-AS1 in the resected tumors in G . ( n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001, NS not significant.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Quantitative RT-PCR, Transfection, Control, shRNA, Incubation, Injection, Expressing, Stable Transfection

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A qRT-PCR analysis of TM4SF1-AS1 in the indicated cellular fractions from HSC-45 cells. U6 snRNA and ACTB served as endogenous controls. ( n = 3). B Association between TM4SF1-AS1 and Pur-α in the indicated subcellular fractions from HSC-45 cells. Pur-α was detected by western blotting in proteins pulled-down with BrU-labeled TM4SF1-AS1, antisense of TM4SF1-AS1 or beads. C Association between Pur-α and YB-1. Immunoprecipitated YB-1 or Pur-α from HSC-45 cell extracts were probed for co-precipitating proteins. D YB-1 was immunoprecipitated from HSC-45 cells with or without RNase A treatment, after which Pur-α was detected by western blotting. E YB-1 or Pur-α in the indicated subcellular fractions from HSC-45 cells were immunoprecipitated with or without RNase A treatment and then probed for co-precipitating proteins. F Results of RIP assays. Pur-α (upper) or YB-1 (lower) was immunoprecipitated from HSC-45 cells, after which co-precipitated TM4SF1-AS1 was detected with qRT-PCR. IgG served as a negative control. ( n = 3). G qRT-PCR analysis of Pur-α (upper) and YB-1 (lower) in HSC-45 cells transfected with the indicated siRNAs. ( n = 3). H Results of cell viability assays with HSC-45 cells transfected with the indicated siRNAs. ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Quantitative RT-PCR, Western Blot, Labeling, Immunoprecipitation, Negative Control, Transfection

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A Heatmap showing expression of genes suppressed by TM4SF1-AS1 knockdown in HSC-45 cells. B GO analysis using the genes in A . C qRT-PCR analysis of representative IRDS genes in HSC-45 cells transfected with the indicated siRNAs. ( n = 3). D GSEA analysis using the microarray data. Gene sets with an FDR < 0.01 are shown on the left. Enrichment plots of indicated gene sets are shown on the right. E Western blot analysis of total and phosphorylated STAT1 in HSC-45 cells transfected with the indicated siRNAs. (F, G) qRT-PCR analysis of interferon genes in HSC-45 cells transfected with the indicated siRNAs ( F ) or in SNU638 cells stably transfected with the indicated genes ( G ). ( n = 3). H Western blot analysis of total and phosphorylated STAT1 in SNU638, SNU638-GFP, and SNU638-TM4SF1-AS1 cells. I qRT-PCR analysis of IRDS genes in SNU638-GFP and SNU638-TM4SF1-AS1 cells. ( n = 3). J Western blot analysis of STAT1 and Pur-α in SNU638-TM4SF1-AS1 cells transfected with the indicated siRNAs targeting Pur-α. K qRT-PCR analysis of IRDS genes in SNU638-TM4SF1-AS1 cells transfected with the indicated siRNAs. ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Transfection, Microarray, Western Blot, Stable Transfection

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A Workflow of ChIRP coupled with mass spectrometry (ChIRP-MS) or RNA-seq (ChIRP-RNA-seq) to identify molecules associated with TM4SF1-AS1. B qRT-PCR confirming enrichment of TM4SF1-AS1 in ChIRP products derived from SNU638-TM4SF1-AS1 cells. ( n = 3). C Protein-protein interaction (PPI) network among the proteins identified by ChIRP-MS. Functional categories of the proteins are indicated by node colors. D RIP-qPCR assays validating the ChIRP-MS results. The indicated proteins in HSC-45 cells were immunoprecipitated, and co-precipitated TM4SF1-AS1 was detected by qRT-PCR. IgG served as a negative control. ( n = 3). E Immunofluorescence images showing staining of the SG marker G3PB2 in SNU638, SNU638-GFP, and SNU638-TM4SF1-AS1 cells treated with or without sodium arsenite (SA). Summarized results are shown on the right ( n = 5). Scale bars = 10 μm. F Immunofluorescence indicating G3BP2 (green), TIA1 (white), and inducible MS2-tagged TM4SF1-AS1 (red) in SNU638 cells. Cells were transfected with a MS2 coat protein (MCP)-RFP plasmid and incubated for 8 days with or without Dox. Magnified views of the respective markers are shown on the right. Scale bars = 10 μm. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Mass Spectrometry, RNA Sequencing Assay, Quantitative RT-PCR, Derivative Assay, Functional Assay, Immunoprecipitation, Negative Control, Immunofluorescence, Staining, Marker, Transfection, Plasmid Preparation, Incubation

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A Immunofluorescence indicating RACK1 (green), G3BP1 (white), and inducible MS2-tagged TM4SF1-AS1 (red) in SNU638 cells. The cells were transfected with a MS2 coat protein (MCP)-RFP plasmid and incubated for 8 days with or without Dox. Magnified views of the respective markers are shown on the right. Scale bars = 10 μm. B Immunofluorescence images of G3PB2 in HSC-45 cells incubated for 8 days with or without Dox and expressing the indicated inducible shRNAs. Representative images are shown on the left. Summarized results are on the right. ( n = 5). C , D Apoptosis ( C ) and cell cycle ( D ) assays in HSC-45 cells incubated with or without Dox for 8 days and expressing the indicated inducible shRNAs. Representative results are shown on the left. Summarized results are shown on the right. ( n = 3). E , F Western blot analysis of PARP, caspase-3 ( E ) and p38 ( F ) in HSC-45 cells incubated with or without Dox for 8 days and expressing the indicated inducible shRNAs. Cleaved PARP and cleaved caspase-3 are indicated by arrows ( E ). Phosphorylated p38 is indicated by an arrow ( F ). ** P < 0.01, *** P < 0.001.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Incubation, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: A , B Immunofluorescence images showing Pur-α (A) or YB-1 (B), TIA1 and inducible MS2-tagged TM4SF1-AS1 in SNU638 cells incubated for 8 days with or without Dox. Scale bars = 10 μm. C Immunofluorescent staining of G3PB2 in SNU638-TM4SF1-AS1 cells transfected with the indicated siRNAs. Representative results are shown on the left. Summarized results are shown on the right. ( n = 5). Scale bars = 10 μm. D Localization of GFP-tagged Pur-α or YB-1, G3BP1 and TIA1 immunofluorescence in SNU638 cells. Cells were transfected with vectors encoding GFP (upper), GFP-tagged Pur-α (middle) or GFP-tagged YB1 (bottom). Magnified views of the respective markers are shown on the right. Scale bars = 10 μm. E Cell cycle analysis of HSC-45 cells expressing the indicated siRNAs. ( n = 3). F , G Western blot analysis of PARP and caspase-3 ( F ) and total and phosphorylated p38 ( G ) in HSC-45 cells transfected with the indicated siRNAs. Cleaved PARP, cleaved caspase-3 and phosphorylated p38 are indicated by arrows. ** P < 0.01, *** P < 0.001.

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Immunofluorescence, Incubation, Staining, Transfection, Cell Cycle Assay, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

doi: 10.1038/s41419-023-05953-3

Figure Lengend Snippet: In the early steps of gastric tumorigenesis, TM4SF1-AS1 is transcriptionally activated. TM4SF1-AS1 sequesters RACK1 within SGs, which leads to suppression of stress-responsive MAPK signaling and inhibition of apoptosis (created with BioRender.com).

Article Snippet: pCMV6 encoding GFP-fused TM4SF1 was purchased from OriGene Technologies (Rockville, MD, USA). pIDTSMART-AMP encoding TM4SF1-AS1 (ENST496491.1) was purchased from Codex DNA Inc. (San Diego, CA, USA).

Techniques: Inhibition

Journal: Redox Biology

Article Title: Hypoxic acclimation improves cardiac redox homeostasis and protects heart against ischemia-reperfusion injury through upregulation of O-GlcNAcylation

doi: 10.1016/j.redox.2021.101994

Figure Lengend Snippet: HA-induced inflammation stimulated protein O-GlcNAcylation in hearts. A , Immunoblot analysis of IL-1β and IL-6 in the Control and HA hearts (n = 9 per group). B , Immunoblot analysis of IL-1β and IL-6 in the H9c2 cells (IL-1β: n = 7 per group; IL-6: n = 8 per group). C-D , The linear regression analysis between the expression of IL-1β ( C ) or IL-6 ( D ) and protein O-GlcNAcylation level. E-F , Representative blots of protein O-GlcNAc modification in H9c2 cells treated with increasing concentration of IL-1β ( E ) or IL-6 ( F ). G , The glucose uptake rate measure by 2-NBDG in H9c2 cells. H , Immunoblot analysis of protein O-GlcNAc modification in H9c2 cells after HA and neutralizing antibody treatments (n = 4 per group). I , Immunoblot analysis of protein O-GlcNAc modification in H9c2 cells with indicated treatments (n = 7 per group). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01 for indicated comparisons.

Article Snippet: To attenuate protein O-GlcNAcylation, the H9c2 cells were exposed to 6-Diazo-5-oxo-L-nor-Leucine (DON, 20 μM; MCE) for 24 h.

Techniques: Western Blot, Control, Expressing, Modification, Concentration Assay

Journal: Redox Biology

Article Title: Hypoxic acclimation improves cardiac redox homeostasis and protects heart against ischemia-reperfusion injury through upregulation of O-GlcNAcylation

doi: 10.1016/j.redox.2021.101994

Figure Lengend Snippet: PPP was enhanced via O-GlcNAcylation-mediated activation of G6PDH. A , Representative blots of G6PDH in cardiac tissues and H9c2 cells (n = 4 per group). B–C , Representative blots of O-GlcNAc modified G6PDH ( B ) and G6PDH activity ( C ) in H9c2 cells treated with IL-1β for 4 or 24 h (n = 6 per group). D-E , Representative blots of O-GlcNAc modification ( D ) and the G6PDH activity ( E ) in H9c2 cells treated with IL-6 for 4 or 24 h (n = 6 per group). F , The compounds used in this study for targeting HBP pathway enzymes. G-I , Representative blots of O-GlcNAc-modified G6PDH ( G ), G6PDH activity ( H ), and NADPH/NADP + ratio ( I ) in H9c2 cells with indicated treatments (n = 4–5 per group). J , Representative blots of O-GlcNAc modified G6PDH in the mouse hearts with indicated treatments (n = 3 per group). K-M , The G6PDH activity ( K ), NADPH/NADP + ratio ( L ), and GSH/GSSG ratio ( M ) in the mouse hearts with indicated treatments (n = 4–6 per group). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 for indicated comparisons. NS: no significant difference.

Article Snippet: To attenuate protein O-GlcNAcylation, the H9c2 cells were exposed to 6-Diazo-5-oxo-L-nor-Leucine (DON, 20 μM; MCE) for 24 h.

Techniques: Activation Assay, Modification, Activity Assay

Journal: Redox Biology

Article Title: Hypoxic acclimation improves cardiac redox homeostasis and protects heart against ischemia-reperfusion injury through upregulation of O-GlcNAcylation

doi: 10.1016/j.redox.2021.101994

Figure Lengend Snippet: HA attenuated acute oxidative insult in H9c2 cells in an O-GlcNAcylation-dependent manner. A , The global cellular reactive oxygen species (ROS) production measured in H9c2 cells measured by CM-H2DCFDA (n = 6–7 per group). B , Representative fluorescence images of oxidative DNA damage measured by immunostaining for 8-Oxo-dG (red), nuclei (DAPI; blue) and mitochondria (Tom20; green). Scale bar, 10 μm. C , Quantitative analysis of 8-Oxo-dG positive staining in nuclei and mitochondria, and the results were normalized to the intensity in nuclear compartment of the control group (n = 50 cells per group). D , The MitoSOX flow cytometry analysis and the fluorescence intensity in H9c2 cells with indicated treatments (n = 3 per group). E , The oxygen consumption rates (OCR) of H9c2 cells at baseline and after FCCP stimulation (n = 5–6 per group). F , The levels of apoptosis in H9c2 cells with indicated treatments (n = 5 per group). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 for indicated comparisons. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: To attenuate protein O-GlcNAcylation, the H9c2 cells were exposed to 6-Diazo-5-oxo-L-nor-Leucine (DON, 20 μM; MCE) for 24 h.

Techniques: Fluorescence, Immunostaining, Staining, Control, Flow Cytometry

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