Journal: bioRxiv

Article Title: Bioactive coatings on 3D printed scaffolds for bone regeneration: Translation from in vitro to in vivo models and the impact of material properties and growth factor concentration

doi: 10.1101/2023.10.22.560309

Figure Lengend Snippet: HBMSC viability and live (green)/dead (red) cell labelling on uncoated, ELP, PEA/FN/BMP-2 and ELP/PEA/FN/BMP-2 coated PCL-TMA scaffolds. (A) alamarBlue™ HS fluorescence results of ELP, PEA/FN/BMP-2 and ELP/PEA/FN/BMP-2 coated PCL-TMA scaffolds show no significant difference between uncoated PCL-TMA and coated scaffolds, however all coating variations showed a significantly increased fluorescence result at day 14 compared to uncoated PCL-TMA. 2-way ANOVA with Dunnett’s multiple comparisons test, n=3, mean and S.D. shown, ns; non-significant, ****p<0.001. (B) The cell number and location of cells adhered to the scaffold initially at day 1 and subsequent increase in cell coverage at day 14, as seen by fluorescent labelling of cells. Scale bar 1 mm.

Article Snippet: Reagents were purchased as follows: Ethyl acrylate (Sigma, UK), ELP with statherin sequence (SN A 15) (Technical Proteins Nanobiotechnology, Valladolid, Spain); collagenase (Gibco, UK); human fibronectin and human recombinant BMP-2 (R&D systems, Biotechne, UK); recombinant human BMP-2 (Infuse/InductOS® Bone graft kit, Medtronic, USA); alcian blue 8X, light green SF, orange G 85% pure, Paraformaldehyde 96% extra pure, phosphomolybdic acid hydrate 80% (Acros Organics); Picrosirius Red, Van Gieson’s stain, Weigert’s Haematoxylin Parts 1 and 2 (Clintech Ltd, UK); Benzoyl peroxide, GMA solution B, JB4 solution A (Polysciences); GoTaq qPCR master mix, Herring sperm DNA, RNeasy mini prep RNA extraction kit (Promega); phosphate buffered saline (PBS), trypsin/ ethylenediaminetetraacetic acid (EDTA), Dulbecco’s Modified Eagle Medium (DMEM), Alpha Minimum Essential Medium (αMEM), penicillin-streptomycin (Scientific Laboratory Supplies, SLS); 4-Nitrophenol solution 10 nM, acetic acid, acetone, acid fushsin, alizarin red S, alkaline buffer solution, ascorbic acid-2-phosphate, beta-glycerophosphate disodium hydrate salt (βGP), cell lytic M, dexamethasone, fast violet B salts, glycine, histowax, hydrochloric acid, iodoacetamide, ipegal, L-glutamic acid, Naphthol AS-MX phosphate 0.25%, parafilm, PBS (with CaCl 2 /MgCl 2 ), phenyl methyl sulphonyl fluoride, phosphatase substrate, polysorbate 80, ponceau xylidine, silver nitrate, sodium chloride, sodium hydroxide pellets, sucrose, TRIS-EDTA (TE) buffer solution (Merck, UK); Embedding capsule (TAAB Laboratories equipment); alamarBlue™ HS Cell Viability Reagent, 70 µM cell strainer, dibutyl phthalate xylene (DPX), ethidium homodimer-1, fetal calf serum (FCS), fisherbrand grade 01 cellulose general purpose filter paper, Histoclear, isopropanol, methyl benzoate, Quanti-IT™ Picogreen™ ds DNA reagent, Taqman® Reverse Transcription Kit, Vybrant™ CFDA SE Cell Tracer Kit (Thermofisher Scientific, UK); Fast green and sodium thiosulphate (VWR); Lubrithal (Dechra, UK), Isoflurane (Dechra, UK), Buprenorphine (Buprecare® multidose, Animalcare, UK) and Vetasept® sourced from MWI animal health, UK.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Bioactive coatings on 3D printed scaffolds for bone regeneration: Translation from in vitro to in vivo models and the impact of material properties and growth factor concentration

doi: 10.1101/2023.10.22.560309

Figure Lengend Snippet: Assessment of HBMSC differentiation on coated 3D scaffold materials. (A) ALP specific activity of HBMSCs on ELP, PEA/FN/BMP-2 and ELP/PEA/FN/BMP-2 coated PCL-TMA scaffolds at day 7. There was no significant difference between the uncoated PCL-TMA and the coatings of interest in basal or osteogenic conditions. (B) ALP specific activity results comparing 100 ng/mL and 5 µg/mL BMP-2 coating solution concentration at day 7. There was a significant increase in ALP production by HBMSCs in response to the 5 μg/mL BMP-2 coating solution compared to the 100 ng/mL concentration BMP-2 solution in osteogenic culture conditions only. (C) ALP gene expression at day 7 was significantly enhanced for ELP coating than uncoated nylon in basal culture conditions, with the PEA/FN/BMP-2, ELP/PEA/FN/BMP-2 coatings displaying significantly reduced ALP gene expression in osteogenic conditions. (D) Collagen1A1 gene expression at day 7 was not significantly greater than uncoated nylon for any of the coatings in basal or osteogenic media conditions. 2-way ANOVA with Dunnett’s multiple comparisons test, n=3, mean and S.D. shown, ns; non-significant, *p<0.05, **p<0.01, ****p=<0.0001. (E) Alizarin red staining at day 27 indicated red stain uptake in the ELP coated and mineralised scaffolds when no cells were seeded, due to the constituents of the coatings themselves. ELP coating alone and with subsequent mineralisation, lead to enhanced staining due to mineral deposition on the surface of the scaffold in osteogenic culture conditions. Representative images shown, n=3, scale bar 1 mm.

Article Snippet: Reagents were purchased as follows: Ethyl acrylate (Sigma, UK), ELP with statherin sequence (SN A 15) (Technical Proteins Nanobiotechnology, Valladolid, Spain); collagenase (Gibco, UK); human fibronectin and human recombinant BMP-2 (R&D systems, Biotechne, UK); recombinant human BMP-2 (Infuse/InductOS® Bone graft kit, Medtronic, USA); alcian blue 8X, light green SF, orange G 85% pure, Paraformaldehyde 96% extra pure, phosphomolybdic acid hydrate 80% (Acros Organics); Picrosirius Red, Van Gieson’s stain, Weigert’s Haematoxylin Parts 1 and 2 (Clintech Ltd, UK); Benzoyl peroxide, GMA solution B, JB4 solution A (Polysciences); GoTaq qPCR master mix, Herring sperm DNA, RNeasy mini prep RNA extraction kit (Promega); phosphate buffered saline (PBS), trypsin/ ethylenediaminetetraacetic acid (EDTA), Dulbecco’s Modified Eagle Medium (DMEM), Alpha Minimum Essential Medium (αMEM), penicillin-streptomycin (Scientific Laboratory Supplies, SLS); 4-Nitrophenol solution 10 nM, acetic acid, acetone, acid fushsin, alizarin red S, alkaline buffer solution, ascorbic acid-2-phosphate, beta-glycerophosphate disodium hydrate salt (βGP), cell lytic M, dexamethasone, fast violet B salts, glycine, histowax, hydrochloric acid, iodoacetamide, ipegal, L-glutamic acid, Naphthol AS-MX phosphate 0.25%, parafilm, PBS (with CaCl 2 /MgCl 2 ), phenyl methyl sulphonyl fluoride, phosphatase substrate, polysorbate 80, ponceau xylidine, silver nitrate, sodium chloride, sodium hydroxide pellets, sucrose, TRIS-EDTA (TE) buffer solution (Merck, UK); Embedding capsule (TAAB Laboratories equipment); alamarBlue™ HS Cell Viability Reagent, 70 µM cell strainer, dibutyl phthalate xylene (DPX), ethidium homodimer-1, fetal calf serum (FCS), fisherbrand grade 01 cellulose general purpose filter paper, Histoclear, isopropanol, methyl benzoate, Quanti-IT™ Picogreen™ ds DNA reagent, Taqman® Reverse Transcription Kit, Vybrant™ CFDA SE Cell Tracer Kit (Thermofisher Scientific, UK); Fast green and sodium thiosulphate (VWR); Lubrithal (Dechra, UK), Isoflurane (Dechra, UK), Buprenorphine (Buprecare® multidose, Animalcare, UK) and Vetasept® sourced from MWI animal health, UK.

Techniques: Activity Assay, Concentration Assay, Expressing, Staining

Journal: bioRxiv

Article Title: Bioactive coatings on 3D printed scaffolds for bone regeneration: Translation from in vitro to in vivo models and the impact of material properties and growth factor concentration

doi: 10.1101/2023.10.22.560309

Figure Lengend Snippet: CAM assay viability and Chalkley score results for PCL-TMA scaffolds. (A) Chick viability was suboptimal due to poor chick development, n=6. (B) There was no significant difference in Chalkley score between uncoated PCL-TMA and the coated scaffolds, (uncoated n=4, ELP n=4, PEA/FN/BMP-2 n=4, ELP/PEA/FN/BMP-2 n=3), ns=non-significant. One-way ANOVA with Dunnett’s multiple comparisons test was used for statistical analysis, mean and S.D. shown. (C) Photographs of representative uncoated PCL-TMA and ELP coating, PEA/FN/BMP-2 and ELP/PEA/FN/BMP-2 coated PCL-TMA scaffolds on the CAM. The PCL-TMA material, the ELP and PEA/FN/BMP-2 coatings were biocompatible and supported angiogenesis. Scale bar 5 mm. (D) Histological staining (Alcian blue and Sirius red or Goldner’s trichrome) of PCL-TMA scaffolds surrounded by CAM tissue. The PCL-TMA scaffold material did not support sectioning, with fragments remaining (arrow), but tissue around the prior scaffold (*) could be determined. Scale bar 100 μm.

Article Snippet: Reagents were purchased as follows: Ethyl acrylate (Sigma, UK), ELP with statherin sequence (SN A 15) (Technical Proteins Nanobiotechnology, Valladolid, Spain); collagenase (Gibco, UK); human fibronectin and human recombinant BMP-2 (R&D systems, Biotechne, UK); recombinant human BMP-2 (Infuse/InductOS® Bone graft kit, Medtronic, USA); alcian blue 8X, light green SF, orange G 85% pure, Paraformaldehyde 96% extra pure, phosphomolybdic acid hydrate 80% (Acros Organics); Picrosirius Red, Van Gieson’s stain, Weigert’s Haematoxylin Parts 1 and 2 (Clintech Ltd, UK); Benzoyl peroxide, GMA solution B, JB4 solution A (Polysciences); GoTaq qPCR master mix, Herring sperm DNA, RNeasy mini prep RNA extraction kit (Promega); phosphate buffered saline (PBS), trypsin/ ethylenediaminetetraacetic acid (EDTA), Dulbecco’s Modified Eagle Medium (DMEM), Alpha Minimum Essential Medium (αMEM), penicillin-streptomycin (Scientific Laboratory Supplies, SLS); 4-Nitrophenol solution 10 nM, acetic acid, acetone, acid fushsin, alizarin red S, alkaline buffer solution, ascorbic acid-2-phosphate, beta-glycerophosphate disodium hydrate salt (βGP), cell lytic M, dexamethasone, fast violet B salts, glycine, histowax, hydrochloric acid, iodoacetamide, ipegal, L-glutamic acid, Naphthol AS-MX phosphate 0.25%, parafilm, PBS (with CaCl 2 /MgCl 2 ), phenyl methyl sulphonyl fluoride, phosphatase substrate, polysorbate 80, ponceau xylidine, silver nitrate, sodium chloride, sodium hydroxide pellets, sucrose, TRIS-EDTA (TE) buffer solution (Merck, UK); Embedding capsule (TAAB Laboratories equipment); alamarBlue™ HS Cell Viability Reagent, 70 µM cell strainer, dibutyl phthalate xylene (DPX), ethidium homodimer-1, fetal calf serum (FCS), fisherbrand grade 01 cellulose general purpose filter paper, Histoclear, isopropanol, methyl benzoate, Quanti-IT™ Picogreen™ ds DNA reagent, Taqman® Reverse Transcription Kit, Vybrant™ CFDA SE Cell Tracer Kit (Thermofisher Scientific, UK); Fast green and sodium thiosulphate (VWR); Lubrithal (Dechra, UK), Isoflurane (Dechra, UK), Buprenorphine (Buprecare® multidose, Animalcare, UK) and Vetasept® sourced from MWI animal health, UK.

Techniques: Chick Chorioallantoic Membrane Assay, Staining

Journal: bioRxiv

Article Title: Bioactive coatings on 3D printed scaffolds for bone regeneration: Translation from in vitro to in vivo models and the impact of material properties and growth factor concentration

doi: 10.1101/2023.10.22.560309

Figure Lengend Snippet: µCT results of the murine subcutaneous implantation study. (A) Representative µCT images with no bone formation observed in uncoated, PEA/FN/BMP-2, ELP, or ELP/PEA/FN/BMP-2 coated PCL-TMA scaffolds, however the collagen sponge with 5 µg of BMP-2 showed mineralisation in all mice (within red circles) at week 6. (B) Quantification of bone volume formed in the mouse subcutaneous implant model using PCL-TMA scaffolds and collagen sponge/BMP-2. (i) The collagen sponge displayed significant bone formation at 8 weeks compared to the uncoated PCL-TMA scaffold. (ii) There was no significant difference between the negligible bone formation on the coated scaffolds compared to the uncoated PCL-TMA scaffolds. One-way ANOVA with Dunnett’s multiple comparisons test, ns= not significant, ****p<0.0001. N=9 uncoated scaffolds, n=9 collagen sponge, n=6 PEA/FN/BMP-2 and n=6 ELP/PEA/FN/BMP-2 coated scaffolds, mean and S.D. shown.

Article Snippet: Reagents were purchased as follows: Ethyl acrylate (Sigma, UK), ELP with statherin sequence (SN A 15) (Technical Proteins Nanobiotechnology, Valladolid, Spain); collagenase (Gibco, UK); human fibronectin and human recombinant BMP-2 (R&D systems, Biotechne, UK); recombinant human BMP-2 (Infuse/InductOS® Bone graft kit, Medtronic, USA); alcian blue 8X, light green SF, orange G 85% pure, Paraformaldehyde 96% extra pure, phosphomolybdic acid hydrate 80% (Acros Organics); Picrosirius Red, Van Gieson’s stain, Weigert’s Haematoxylin Parts 1 and 2 (Clintech Ltd, UK); Benzoyl peroxide, GMA solution B, JB4 solution A (Polysciences); GoTaq qPCR master mix, Herring sperm DNA, RNeasy mini prep RNA extraction kit (Promega); phosphate buffered saline (PBS), trypsin/ ethylenediaminetetraacetic acid (EDTA), Dulbecco’s Modified Eagle Medium (DMEM), Alpha Minimum Essential Medium (αMEM), penicillin-streptomycin (Scientific Laboratory Supplies, SLS); 4-Nitrophenol solution 10 nM, acetic acid, acetone, acid fushsin, alizarin red S, alkaline buffer solution, ascorbic acid-2-phosphate, beta-glycerophosphate disodium hydrate salt (βGP), cell lytic M, dexamethasone, fast violet B salts, glycine, histowax, hydrochloric acid, iodoacetamide, ipegal, L-glutamic acid, Naphthol AS-MX phosphate 0.25%, parafilm, PBS (with CaCl 2 /MgCl 2 ), phenyl methyl sulphonyl fluoride, phosphatase substrate, polysorbate 80, ponceau xylidine, silver nitrate, sodium chloride, sodium hydroxide pellets, sucrose, TRIS-EDTA (TE) buffer solution (Merck, UK); Embedding capsule (TAAB Laboratories equipment); alamarBlue™ HS Cell Viability Reagent, 70 µM cell strainer, dibutyl phthalate xylene (DPX), ethidium homodimer-1, fetal calf serum (FCS), fisherbrand grade 01 cellulose general purpose filter paper, Histoclear, isopropanol, methyl benzoate, Quanti-IT™ Picogreen™ ds DNA reagent, Taqman® Reverse Transcription Kit, Vybrant™ CFDA SE Cell Tracer Kit (Thermofisher Scientific, UK); Fast green and sodium thiosulphate (VWR); Lubrithal (Dechra, UK), Isoflurane (Dechra, UK), Buprenorphine (Buprecare® multidose, Animalcare, UK) and Vetasept® sourced from MWI animal health, UK.

Techniques:

Journal: bioRxiv

Article Title: Bioactive coatings on 3D printed scaffolds for bone regeneration: Translation from in vitro to in vivo models and the impact of material properties and growth factor concentration

doi: 10.1101/2023.10.22.560309

Figure Lengend Snippet: Alcian blue and Sirius red, Goldner’s trichrome, Alizarin red and Von Kossa staining of PCL-TMA scaffolds and collagen sponge/BMP-2. The scaffolds were not amenable to sectioning; however, the surrounding tissue remained. (*) PCL-TMA scaffold area or collagen sponge/BMP-2. (A) Shards of PCL-TMA material (black arrow) remain in the section. (B) Vivid red staining muscle was seen but no bone formation. (C and D) No bone formation was found on the uncoated scaffold. (E - H) Only the collagen sponge displayed marked mineralisation and bone formation around the periphery (arrows). (I - T) No bone formation was seen on the ELP, PEA/FN/BMP-2 or ELP/PEA/FN/BMP-2 coated scaffolds, with the ridges of the scaffold material seen surrounded by tissue (J arrow). Scale bar 100 μm.

Article Snippet: Reagents were purchased as follows: Ethyl acrylate (Sigma, UK), ELP with statherin sequence (SN A 15) (Technical Proteins Nanobiotechnology, Valladolid, Spain); collagenase (Gibco, UK); human fibronectin and human recombinant BMP-2 (R&D systems, Biotechne, UK); recombinant human BMP-2 (Infuse/InductOS® Bone graft kit, Medtronic, USA); alcian blue 8X, light green SF, orange G 85% pure, Paraformaldehyde 96% extra pure, phosphomolybdic acid hydrate 80% (Acros Organics); Picrosirius Red, Van Gieson’s stain, Weigert’s Haematoxylin Parts 1 and 2 (Clintech Ltd, UK); Benzoyl peroxide, GMA solution B, JB4 solution A (Polysciences); GoTaq qPCR master mix, Herring sperm DNA, RNeasy mini prep RNA extraction kit (Promega); phosphate buffered saline (PBS), trypsin/ ethylenediaminetetraacetic acid (EDTA), Dulbecco’s Modified Eagle Medium (DMEM), Alpha Minimum Essential Medium (αMEM), penicillin-streptomycin (Scientific Laboratory Supplies, SLS); 4-Nitrophenol solution 10 nM, acetic acid, acetone, acid fushsin, alizarin red S, alkaline buffer solution, ascorbic acid-2-phosphate, beta-glycerophosphate disodium hydrate salt (βGP), cell lytic M, dexamethasone, fast violet B salts, glycine, histowax, hydrochloric acid, iodoacetamide, ipegal, L-glutamic acid, Naphthol AS-MX phosphate 0.25%, parafilm, PBS (with CaCl 2 /MgCl 2 ), phenyl methyl sulphonyl fluoride, phosphatase substrate, polysorbate 80, ponceau xylidine, silver nitrate, sodium chloride, sodium hydroxide pellets, sucrose, TRIS-EDTA (TE) buffer solution (Merck, UK); Embedding capsule (TAAB Laboratories equipment); alamarBlue™ HS Cell Viability Reagent, 70 µM cell strainer, dibutyl phthalate xylene (DPX), ethidium homodimer-1, fetal calf serum (FCS), fisherbrand grade 01 cellulose general purpose filter paper, Histoclear, isopropanol, methyl benzoate, Quanti-IT™ Picogreen™ ds DNA reagent, Taqman® Reverse Transcription Kit, Vybrant™ CFDA SE Cell Tracer Kit (Thermofisher Scientific, UK); Fast green and sodium thiosulphate (VWR); Lubrithal (Dechra, UK), Isoflurane (Dechra, UK), Buprenorphine (Buprecare® multidose, Animalcare, UK) and Vetasept® sourced from MWI animal health, UK.

Techniques: Staining

Journal: Cancer

Article Title: Colorectal carcinoma cell production of transforming growth factor beta decreases expression of endothelial cell vascular endothelial growth factor receptor 2.

doi: 10.1002/cncr.26247

Figure Lengend Snippet: Figure 6. (A) Representative Western blot shows vascular endothelial growth factor receptor 2 (VEGFR2) and activated Smad protein expression and (B) proportion of VEGFR2 protein levels in vehicle-treated and 5 ng/mL transforming growth factor beta (TGF-b)-treated bovine aortic endothelial cells (BAECs) cultured in atmospheric (21%) O2 and <0.1% O2 (anoxia) over 6 and 24 hours. Analysis of variance, P ¼ .0015; n ¼ 4; *P < .05. (C) VEGFR2 and Smad protein levels in BAECs treated for 24 hours with 0 to 50 ng/mL bone morphogenetic protein 9 (BMP9) are shown. (D) VEGFR2 and Smad protein levels in BAECs treated for 24 hours with combinations of 10 ng/mL BMP9, 5 ng/mL TGF-b1, and 5 lM SB-431542 (SB) are shown. Western blot is representative of 2 independent experiments.

Article Snippet: BAECs were grown until confluent and serum-starved overnight before treatment in serum-free medium with 0.1 to 20 ng/mL of recombinant human TGF-b1; 10 ng/mL recombinant human BMP9 (both R&D Systems, Minneapolis, Minn); 5 lM SB431542, an inhibitor of TGbR-I isoforms ALK5, ALK4, and ALK722; and 2.5 lM SD-208, an inhibitor of ALK5 and ALK4 (both Tocris Bioscience, Ellisville, Mo).23 CRC cells were grown until superconfluent, serumstarved overnight, then incubated in fresh serum-free DMEM for 48 hours.

Techniques: Western Blot, Expressing, Cell Culture

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: Sequences used in this study.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques:

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: The expression of lncRNA BMPR1B-AS1 in endometrial cancer tissues and cell lines. A, Volcano map of dysregulated lncRNAs between EC tissues and adjacent normal tissues. The results were obtained from TCGA database. B, Expression levels of lncRNA BMPR1B-AS1 in paired EC and adjacent noncancerous tissues (n = 28). C, RT-qPCR analysis of BMPR1B-AS1 expression in human EC cell lines (Ishikawa, Hec-1a and Hec-1b cells). The data are representative of three independent experiments. Bars, ±SD. **p < 0.01, ****p < 0.0001.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: Overexpression of BMPR1B-AS1 promotes the proliferation, migration and invasion of Hec-1b cells while inhibiting apoptosis. A, Overexpression of BMPR1B-AS1 was confirmed by RT-qPCR. B, Overexpression of BMPR1B-AS1 promoted the proliferation of Hec-1b cells. C, E, G, H, Transwell migration assay (magnification, 200×) and wound healing assay (magnification, 100×) showed that overexpression of BMPR1B-AS1 facilitated the migration of Hec-1b cells. D, F, Transwell invasion assay showed that overexpression of BMPR1B-AS1 facilitated the invasion of Hec-1b cells (magnification, 200×). I, K, Flow cytometry results showed that BMPR1B-AS1 overexpression inhibited Hec-1b cell apoptosis. J, L, Flow cytometry results showed that BMPR1B-AS1 overexpression increased the accumulation of cells in the S-phase and decreased the accumulation of cells in the G0/G1 phase. M, N, O, P, Q, Western blot and analysis showed that the protein level of E-cadherin was decreased, but the protein levels of N-cadherin, Cyclin D1 and CDK4 were increased in the BMPR1B-AS1 overexpression group. Lv, lentiviral vector. The data are representative of three independent experiments. Bars, ±SD. *P < .05, **P < .01, ***P < .001, ****P < 0.0001.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: Over Expression, Migration, Quantitative RT-PCR, Transwell Migration Assay, Wound Healing Assay, Transwell Invasion Assay, Flow Cytometry, Western Blot, Plasmid Preparation

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: Knockdown of BMPR1B-AS1 expression inhibits the proliferation, migration and invasion of Ishikawa cells while promoting apoptosis. A, BMPR1B-AS1 knockdown efficiency was confirmed by RT-qPCR. B, Knockdown of BMPR1B-AS1 expression inhibited the proliferation of Ishikawa cells. C, E, G, H, Transwell migration assay (magnification, 200×) and wound healing assay (magnification, 100×) showed that knockdown of BMPR1B-AS1 expression decreased the migration of Ishikawa cells. D, F, Transwell invasion assay showed that knockdown of BMPR1B-AS1 expression decreased the invasion of Ishikawa cells (magnification, 200×). I, K, Flow cytometry results showed that BMPR1B-AS1 knockdown facilitated Ishikawa cell apoptosis. J, L, Flow cytometry results showed that BMPR1B-AS1 knockdown increased the accumulation of cells in the G0/G1 phase and decreased the accumulation of cells in the S-phase. M, N, O, P, Q, Western blot and analysis showed that the protein level of E-cadherin was increased, but the protein levels of N-cadherin, Cyclin D1 and CDK4 were decreased in the BMPR1B-AS1 knockdown group. Sh, short hairpin. The data are representative of three independent experiments. Bars, ±SD. **P < .01, ***P < .001, ****P < 0.0001.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: Expressing, Migration, Quantitative RT-PCR, Transwell Migration Assay, Wound Healing Assay, Transwell Invasion Assay, Flow Cytometry, Western Blot

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: BMPR1B-AS1 functions as an efficient miR-7-2-3p sponge in Hec-1b and Ishikawa cells. A, FISH assay showed that the intracellular localization of BMPR1B-AS1 was mainly in the cytoplasm in Ishikawa cells. 18S and U6 were used as controls. B, Putative binding sites of miR-7-2-3p and BMPR1B-AS1. C, Negative correlation was found between BMPR1B-AS1 expression and miR-7-2-3p expression among the 28 endometrial cancer tissue specimens. R = −.549, P = .002. D, E, The effect of the miR-7-2-3p mimic on the luciferase activities of 293 T cells transfected with WT or MUT BMPR1B-AS1 was detected 24 h and 48 h after transfection, respectively. F, RT-qPCR results showed that miR-7-2-3p expression was downregulated after BMPR1B-AS1 overexpression in Hec-1b cells. G, RT-qPCR results showed that miR-7-2-3p expression was upregulated after BMPR1B-AS1 knockdown in Ishikawa cells. H, miR-7-2-3p expression was upregulated after transfection with the miR-7-2-3p mimic in Hec-1b cells. I, The miR-7-2-3p mimic reversed the BMPR1B-AS1-mediated downregulation of miR-7-2-3p expression. J, miR-7-2-3p expression was downregulated after transfection of Ishikawa cells with the miR-7-2-3p inhibitor. K, The miR-7-2-3p inhibitor attenuated the BMPR1B-AS1-mediated upregulation of miR-7-2-3p expression. The data are representative of three independent experiments. Bars, ±SD. *P < .05, **P < .01, ***P < .001, ****P < 0.0001.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: Binding Assay, Expressing, Luciferase, Transfection, Quantitative RT-PCR, Over Expression

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: Upregulation of miR-7-2-3p expression reverses the BMPR1B-AS1 overexpression-induced progression of Hec-1b cells. A, CCK8 assay showed that cotransfection with the miR-7-2-3p mimic suppressed the BMPR1B-AS1 overexpression-induced proliferation of Hec-1b cells. B, D, F, G, Transwell migration assay (magnification, 200×) and wound healing assay (magnification, 100×) showed that cotransfection with the miR-7-2-3p mimic suppressed the BMPR1B-AS1 overexpression-induced migration of Hec-1b cells. C, E, Transwell invasion assay (magnification, 200×) showed that cotransfection with the miR-7-2-3p mimic suppressed the BMPR1B-AS1 overexpression-induced invasion of Hec-1b cells. H-K, Flow cytometry showed that cotransfection with the miR-7-2-3p mimic accelerated apoptosis and cell cycle arrest, and these effects were inhibited by BMPR1B-AS1 overexpression in Hec-1b cells. The data are representative of three independent experiments. Bars, ±SD. *P < .05, **P < .01, ***P < .001, ****P < 0.0001.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: Expressing, Over Expression, CCK-8 Assay, Cotransfection, Transwell Migration Assay, Wound Healing Assay, Migration, Transwell Invasion Assay, Flow Cytometry

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: Downregulation of miR-7-2-3p expression reverses the BMPR1B-AS1 knockdown-mediated inhibition of Ishikawa cells. A, CCK8 assay showed that cotransfection with the miR-7-2-3p inhibitor reversed the BMPR1B-AS1 knockdown-mediated inhibition of proliferation of Ishikawa cells. B, D, F, G, Transwell migration assay (magnification, 200×) and wound healing assay (magnification, 100×) showed that cotransfection with the miR-7-2-3p inhibitor reversed the BMPR1B-AS1 knockdown-mediated inhibition of migration of Ishikawa cells. C, E, Transwell invasion assay (magnification, 200×) showed that cotransfection with the miR-7-2-3p inhibitor reversed the BMPR1B-AS1 knockdown-mediated inhibition of invasion of Ishikawa cells. H-K, Flow cytometry showed that cotransfection with the miR-7-2-3p inhibitor inhibited apoptosis and cell cycle arrest, and these effects were enhanced by BMPR1B-AS1 knockdown in Ishikawa cells. The data are representative of three independent experiments. Bars, ±SD. **P < .01, ***P < .001, ****P < 0.0001.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: Expressing, Inhibition, CCK-8 Assay, Cotransfection, Transwell Migration Assay, Wound Healing Assay, Migration, Transwell Invasion Assay, Flow Cytometry

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: DCLK1 is a target gene of miR-7-2-3p, and BMPR1B-AS1 regulates DCLK1 expression by competitively binding to miR-7-2-3p in EC cell lines. A, Putative binding sites of miR-7-2-3p and DCLK1 mRNA. B, C, The effect of the miR-7-2-3p mimic on the luciferase activities of cells transfected with WT or MUT DCLK1 was detected after 24 h and 48 h, respectively. D, Negative correlation was found between miR-7-2-3p expression and DCLK1 mRNA expression among the 28 endometrial cancer tissue specimens. R = −.531, P = .004. E, Positive correlation was found between BMPR1B-AS1 expression and DCLK1 mRNA expression among the 28 endometrial cancer tissue specimens. R = .690, P = .000. F, J, N, RT-qPCR and western blotting results showed that DCLK1 mRNA and protein expression was upregulated after BMPR1B-AS1 overexpression. G, K, O, RT-qPCR and western blotting results showed that DCLK1 mRNA and protein expression was downregulated after BMPR1B-AS1 knockdown. H, L, P, RT-qPCR and western blotting results showed that the miR-7-2-3p mimic attenuated the BMPR1B-AS1-mediated upregulation of DCLK1 mRNA and protein expression. I, M, Q, RT-qPCR and western blotting results showed that the miR-7-2-3p inhibitor reversed the effects of BMPR1B-AS1 knockdown on DCLK1 mRNA and protein expression. The data are representative of three independent experiments. Bars, ±SD. *P < .05, **P < .01, ***P < .001, ****P < 0.0001.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: Expressing, Binding Assay, Luciferase, Transfection, Quantitative RT-PCR, Western Blot, Over Expression

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: BMPR1B-AS1 promotes endometrial cancer cell growth in vivo by targeting miR-7-2-3p. A, Images of tumor xenografts. B, The growth curves of tumor xenografts demonstrated that BMPR1B-AS1 significantly promote tumor growth in vivo , which was reversed by treatment with agomir-7-2-3p. **P < 0.01 vs. Lv-BMPR1B-AS1, ***P < 0.001 vs. Lv-BMPR1B-AS1, ****P < 0.0001 vs. Lv-BMPR1B-AS1, # P < 0.05 vs. Lv-BMPR1B-AS1+ agomiR-7-2-3p. C, The weights of tumor xenografts derived from five groups. D, The expression levels of BMPR1B-AS1 in five groups were examined by RT-qPCR. E, The expression levels of miR-7-2-3p in five groups were examined by RT-qPCR. F, G, The expression levels of DCLK1 in five groups were examined by RT-qPCR and IHC. H, Negative correlation was found between BMPR1B-AS1 and miR-7-2-3p expression in xenografts tissues. R = −.506, P = .000. I, Negative correlation was found between miR-7-2-3p and DCLK1 mRNA expression in xenografts tissues. R = −.452, P = .001. J. Positive correlation was found between BMPR1B-AS1 and DCLK1 mRNA expression in xenografts tissues. R = .387, P = .006. *P < .05, **P < .01, ***P < .001.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: In Vivo, Derivative Assay, Expressing, Quantitative RT-PCR

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: A schematic model of the mechanism by which lncRNA BMPR1B-AS1 regulates endometrial cancer cell malignant behaviors. The BMPR1B-AS1/miR-7-2-3p/DCLK1 axis promotes the progression of endometrial cancer cells via the PI3K/Akt/NF-κB signaling pathway.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: