Journal: bioRxiv

Article Title: Capturing trophectoderm-like stem cells enables step-wisely remodeling of placental development

doi: 10.1101/2025.08.25.672082

Figure Lengend Snippet: (A) The morphology of ESCs, TBLCs and ESCs, TBLCs in TS medium after 3 days of induction. Scale bars, 250 μm. (B) FACS analysis of the percentage of CDX2 + cells from ESCs and TBLCs, as well as ESCs and TBLCs cultured in TS medium, using V6.5 cell line. (C) FACS analysis of the percentage of CD40 + cells from ESCs and TBLCs, as well as ESCs and TBLCs cultured in TS medium, using TC1 cell line. (D) FACS analysis of the percentage of CD40 + TELCs obtained from the TBLCs after induction with different molecules, including FGF4, Activin A, TGFβ1 and BMP4. The corresponding cell morphology is displayed in the lower panel. (E) Scatterplots displaying the transcriptome comparison of TELCs before and after CD40-based FACS using RNA-seq. Upregulated (FC>2) and downregulated (FC<0.5) genes are shown in red and blue, respectively. (F) The morphology of TBLCs of different passages and long-term culture in TX and TS medium, also the morphology of TBLCs after CD40 FACS after induction. Scale bars, 250 μm. (G) Western blotting was used to detect OCT4, CDX2 and EOMES in TELSCs from different passages. β-Tubulin was used as a loading control. (H) The morphology 8C embryos cultured in TX medium. Scale bars, 250 μm. (I) FACS analysis of the percentage of CD40 + cells in TELSC em s at different passages. (J) Immunofluorescence staining of TFAP2C and PEG10 in TBLCs, TELSCs and TELSC em s. Scale bars, 50 μm. (K) Cell cycle analysis of ESCs, TELSCs and TELSC em s. (L) Heatmap indicating the relative expression of TBLCs, TELSCs and TELSC em s. The representative genes and enrichment of GO terms of these genes is shown. (M) Heatmap indicating the relative expression of characteristic genes in TELSCs, TELSC em s and TSCs. Bubble chart showing the relative expression of these genes in mouse embryos. (N) Heatmap indicating the relative expression of characteristic genes in TELSCs, TSCs cultured in TX medium and TSCs cultured in TS medium. Heatmap on the right demonstrating the expression of each cluster in mouse embryos. The representative genes and enrichment of GO terms of these genes is shown. (O) The scatter plot displays differentially expressed genes between TELSCs and TSCs cultured in various media. The bar graph summarizes the number of differentially expressed genes identified under each comparison condition. (P) GSEA analysis of ESCs, TBLCs, TELCs and TELSCs based on “embryonic placenta development” and “placenta development” geneset. (Q) Heatmap indicating the differentially expressed genes in Hippo pathway of TELSCs and TBLCs. (R) Heatmap indicating the relative expression of characteristic genes in TELSCs, TSCs cultured in TX medium and TSCs cultured in TS medium. Bubble chart showing the relative expression of these genes in mouse embryos. (S) Phase contrast images of TBLCs cultured in TS medium for 24h supplemented with Verteporfin at the indicated concentration. Scale bars, 100 µm. (T) Heatmap indicating the differentially expressed genes of TELCs and TBLCs induction in TS medium plus verteporfin. Bubble chart showing the relative expression of these genes in mouse embryos. (U) GSEA analysis of TELCs, TBLCs induction in TS medium and in TS medium plus verteporfin based on TE geneset. (V) The morphology of TELSCs cultured in TS medium, TS medium plus ITS-X and TS medium plus TGFβ1. (W) Heatmap indicating the differentially expressed genes of TELSCs, TBLCs induction in TX medium withdraw ITS-X, in TS medium and in TS medium plus ITS-X. Heatmap on the right demonstrating the expression of each cluster in mouse embryos. The representative genes and enrichment of GO terms of these genes is shown. (X) GSEA analysis of TBLCs induction in TX medium withdraw ITS-X and in TX medium based on “Positive regulation of stem cell proliferation” and “Positive regulation of cell cycle” geneset.

Article Snippet: All TSLs were cultured on Matrigel-coated plates, in 30% TS medium (RPMI 1640 (GIBCO, 11875119), 20% FBS, 1% GlutaMax (GIBCO, 35050061), 1% penicillin-streptomycin (GIBCO, 15140163), 1% sodium pyruvate (GIBCO, 11360070)) and 70% MEF-conditioned TS medium supplemented with 25 ng/ml human recombinant FGF4 (MCE, HY-P7014) and 1 μg/ml heparin (STEMCELL, 7980).

Techniques: Cell Culture, Comparison, RNA Sequencing, Western Blot, Control, Immunofluorescence, Staining, Cell Cycle Assay, Expressing, Concentration Assay

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-2–expressing breast cancer cells mitigate radiation-induced oxidative stress through nitric oxide signaling

doi: 10.1172/JCI181368

Figure Lengend Snippet: ( A ) A Kaplan-Meier curve of overall survival for TNBC patients given radiotherapy and segregated based on median NRP2 mRNA expression from GEO GSE199633 ( n = 55). Gehan-Breslow-Wilcoxon test with * P < 0.05. ( B ) The TNBC cell lines indicated were given a radiation dose of 0, 5, 10 Gy, or 2 Gy × 5 and the percentage of cells with NRP2 surface expression was quantified by flow cytometry ( n = 3). ( C ) Validation of NRP2 knockdown in BT549 and 4T1 cells transfected with shRNAs (shNRP2-1, shNRP2-2) compared with the cells transfected with a control (shCtrl) by immunoblotting. ( D ) Clonogenic assay of BT549 shCtrl, shNRP2-1, and shNRP2-2 cells after irradiation (0–8 Gy; n = 2, representative image). ( E ) Clonogenic assay of BT549 parental cells treated with either hIgG or aNRP2-10 and irradiated (0–8 Gy; n = 2, representative image). ( F ) Clonogenic assay of 4T1 shCtrl, shNRP2-1, and shNRP2-2 cells that had been irradiated (0–8 Gy; n = 2, representative image). ( G ) Clonogenic assay of 4T1 parental cells treated with either hIgG or aNRP2-28 and irradiated (0–8 Gy; n = 2, representative image).* P < 0.05. ( H ) CALYPSO-based analysis of organoid viability after treatment with either hIgG or aNRP2-10 and radiation (10 Gy). Calcein AM is a marker of live cells, and propidium iodide is a marker for dead cells. Scale bars: 100 μm. The bar graph shows the viability measurement for 10 organoids in each condition 48 hours after irradiation. ** P < 0.01. ( I ) Viability of a PDX sorted for NRP2 hi and NPR2 lo expression and then treated with either aNRP2-10 or hIgG prior to irradiation (0 Gy or 10 Gy) was assessed 48 hours after irradiation ( n = 2). Data are presented as means ± SD ( B – I ). Statistical analysis was performed using 2-tailed Student’s t test ( H ) or 2-way ANOVA multiple comparisons ( D – G and I ). ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The following antibodies were used for immunoblotting: tubulin (Cell Signaling Technology, 3873), β-actin (Cell Signaling Technology, 3700S), GAPDH (14C10) (Cell Signaling Technology, 2118S), human NRP2 (aNRP2-36v2 obtained from aTyr; ref. ), mouse NRP2 (R&D Systems, AF2215), human NOS2 (Cell Signaling Technology, 39898), mouse NOS2 (D6B6S) (Cell Signaling Technology, 13120), nitrotyrosine antibody (Santa Cruz Biotechnology, sc-32757), Gli1 (Cell Signaling Technology, 2553s), KEAP1 (D6B12) (Cell Signaling Technology, 8047s), and phospho-histone H2A.X (Ser139) (20E3) (Cell Signaling Technology, 9718s).

Techniques: Expressing, Flow Cytometry, Knockdown, Transfection, Control, Western Blot, Clonogenic Assay, Irradiation, Marker

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-2–expressing breast cancer cells mitigate radiation-induced oxidative stress through nitric oxide signaling

doi: 10.1172/JCI181368

Figure Lengend Snippet: ( A ) The enrichment score associated with the nitric oxide–mediated signal transduction gene set from gene ontology biological pathways (GOBP). ( B ) Representative IHC images of a TNBC patient tumor immunostained with antibodies against NRP2, nitrotyrosine, and DAPI. Scale bars: 200 μm. ( C ) NOS2 mRNA and protein expression in control and shNRP2 cells were quantified by qPCR and immunoblotting ( n = 3). *** P < 0.001; **** P < 0.0001. ( D ) NO production in control and shNRP2 was estimated based on the Nitrite Assay Kit ( n = 3). **** P < 0.0001. ( E ) Immunoblots of protein nitrotyrosine obtained from BT549 NRP2 knockdown cells given either control full media (FM), conditioned medium from NRP2 hi cells (CM), or c-PTIO (50 μM) that had been added to conditioned media from NRP2 hi cells (CM + c-PTIO). The conditioned media for the latter conditions was added to the NRP2 -knockdown cells 6 times over the course of 24 hours ( n = 3, representative image). ( F ) Clonogenic assay of BT549 cells in which NRP2 had been knocked down using 2 shRNAs and then transfected with t NOS2 with and without doxycycline and irradiated (0–6 Gy; n = 2, representative image). * P < 0.05 Data are presented as means ± SD ( C , D and F ). Statistical analysis was performed using 1-way ANOVA multiple comparisons ( C and D ) or 2-way ANOVA multiple comparisons ( F ).

Article Snippet: The following antibodies were used for immunoblotting: tubulin (Cell Signaling Technology, 3873), β-actin (Cell Signaling Technology, 3700S), GAPDH (14C10) (Cell Signaling Technology, 2118S), human NRP2 (aNRP2-36v2 obtained from aTyr; ref. ), mouse NRP2 (R&D Systems, AF2215), human NOS2 (Cell Signaling Technology, 39898), mouse NOS2 (D6B6S) (Cell Signaling Technology, 13120), nitrotyrosine antibody (Santa Cruz Biotechnology, sc-32757), Gli1 (Cell Signaling Technology, 2553s), KEAP1 (D6B12) (Cell Signaling Technology, 8047s), and phospho-histone H2A.X (Ser139) (20E3) (Cell Signaling Technology, 9718s).

Techniques: Transduction, Expressing, Control, Western Blot, Nitration, Knockdown, Clonogenic Assay, Transfection, Irradiation

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-2–expressing breast cancer cells mitigate radiation-induced oxidative stress through nitric oxide signaling

doi: 10.1172/JCI181368

Figure Lengend Snippet: ( A ) γ-H2AX foci in BT549 control and NRP2-knockdown cells were quantified by immunofluorescence at the time points indicated after 4 Gy irradiation ( n = 3). Representative images of the foci at the respective time points and conditions. Scale bars: 10 μm. **** P < 0.0001. ( B ) ROS levels in BT549 and 4T1 shCtrl and shNPR2 cells were measured 4 hours after a 4 Gy radiation dose ( n = 3). *** P < 0.001; **** P < 0.0001. ( C ) ROS levels in BT549 and 4T1 cells that had been pretreated with either IgG or aNRP2 for 24 hours were measured 4 hours after 4 Gy irradiation ( n = 3). ** P < 0.01; **** P < 0.0001.( D ) DNA damage was quantified by the olive tail moment using the alkaline comet assay in BT549 shCtrl and BT549 shNRP2-1 cells 4 hours after 4 Gy irradiation, with or without NAC treatment 2 hours prior to radiation ( n = 3). Scale bars: 100 μm. * P < 0.05; ** P < 0.01. ( E ) The impact of NOS2 inhibition with 1400W (50 μM) on ROS levels in BT549 shCtrl and shNRP2 cells 4 hours after 4 Gy irradiation ( n = 3). **** P < 0.0001. ( F ) ROS levels were measured 4 hours after 4 Gy radiation in NRP2-knockdown cells transfected with t NOS2 with and without doxycycline ( n = 3). * P < 0.05. Data are presented as means ± SD ( A – F ). Statistical analysis was performed using 2-tailed Student’s t test ( F ), 1-way ANOVA multiple comparisons ( D ), and 2-way ANOVA multiple comparisons ( A – C and E ).

Article Snippet: The following antibodies were used for immunoblotting: tubulin (Cell Signaling Technology, 3873), β-actin (Cell Signaling Technology, 3700S), GAPDH (14C10) (Cell Signaling Technology, 2118S), human NRP2 (aNRP2-36v2 obtained from aTyr; ref. ), mouse NRP2 (R&D Systems, AF2215), human NOS2 (Cell Signaling Technology, 39898), mouse NOS2 (D6B6S) (Cell Signaling Technology, 13120), nitrotyrosine antibody (Santa Cruz Biotechnology, sc-32757), Gli1 (Cell Signaling Technology, 2553s), KEAP1 (D6B12) (Cell Signaling Technology, 8047s), and phospho-histone H2A.X (Ser139) (20E3) (Cell Signaling Technology, 9718s).

Techniques: Control, Knockdown, Immunofluorescence, Irradiation, Alkaline Single Cell Gel Electrophoresis, Inhibition, Transfection

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-2–expressing breast cancer cells mitigate radiation-induced oxidative stress through nitric oxide signaling

doi: 10.1172/JCI181368

Figure Lengend Snippet: We evaluated the Gli1 mRNA expression in ( A ) BT549 shCtrl and shNRP2 cells ( n = 3), ( B ) 4T1-RR cells that had been treated with either IgG or aNRP2-10 for 24 hours ( n = 3), and ( C ) BT549 cells given a combined treatment of radiation (0, 5, and 10 Gy) with antibody for 24 hours ( n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ( D ) NOS2 mRNA expression was quantified in BT549 cells that had been treated with either DMSO or GANT61 (10 μM) for 24 hours ( n = 3). *** P < 0.001. ( E ) Gli1 and NOS2 mRNA expression was quantified in BT549 shCtrl and shGli1 cells ( n = 3). *** P < 0.001; **** P < 0.0001. ( F ) NOS2 mRNA expression in BT549 shNRP2 cells that had been transfected with either empty vector or a Gli1 -HA construct ( n = 3). The immunoblot shows the protein expression of NOS2, Gli1, and GAPDH in the same cells. *** P < 0.001; **** P < 0.0001. ( G ) Binding of Gli1 on the NOS2 promoter was analyzed using ChIP-qPCR in BT549 cells ( n = 2, representative image). ** P < 0.01. ( H ) NOS2 expression of CRISPR-generated mutations of the Gli1-binding site (Gli1-bind KO1 and KO2) compared with control ( n = 3). ( I ) Clonogenic assay of control (sgCtrl), Gli1-bind KO1, and Gli1-bind KO2 cells that had been irradiated (0–6 Gy; n = 2, representative image). * P < 0.05 Data are presented as means ± SD ( A – G , and I ). Statistical analysis was performed using 2-tailed Student’s t test ( B , D , and F ), 1-way ANOVA multiple comparisons ( A , C , and E ), or 2-way ANOVA multiple comparisons ( I ).

Article Snippet: The following antibodies were used for immunoblotting: tubulin (Cell Signaling Technology, 3873), β-actin (Cell Signaling Technology, 3700S), GAPDH (14C10) (Cell Signaling Technology, 2118S), human NRP2 (aNRP2-36v2 obtained from aTyr; ref. ), mouse NRP2 (R&D Systems, AF2215), human NOS2 (Cell Signaling Technology, 39898), mouse NOS2 (D6B6S) (Cell Signaling Technology, 13120), nitrotyrosine antibody (Santa Cruz Biotechnology, sc-32757), Gli1 (Cell Signaling Technology, 2553s), KEAP1 (D6B12) (Cell Signaling Technology, 8047s), and phospho-histone H2A.X (Ser139) (20E3) (Cell Signaling Technology, 9718s).

Techniques: Expressing, Transfection, Plasmid Preparation, Construct, Western Blot, Binding Assay, CRISPR, Generated, Control, Clonogenic Assay, Irradiation

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-2–expressing breast cancer cells mitigate radiation-induced oxidative stress through nitric oxide signaling

doi: 10.1172/JCI181368

Figure Lengend Snippet: ( A ) Immunofluorescence images of DAPI and NFE2L2 staining in BT549 control and NRP2-knockdown cells with a calculation of the nuclear to cytoplasmic (N/C) ratio of NFE2L2 localization ( n = 3). Scale bars: 10 μm. * P < 0.05; *** P < 0.001. ( B ) Immunofluorescence images of DAPI and NFE2L2 staining in BT549 cells treated with either IgG or aNRP2 for 24 hours with a calculation of the nuclear to cytoplasmic ratio of NFE2L2 ( n = 3). Scale bars: 10 μm. * P < 0.05. ( C ) Expression of NFE2L2 target genes ( SLC7A11 , HMOX1 , and PRDX1 ) in NRP2-knockdown cells was quantified by qPCR ( n = 3).** P < 0.01; *** P < 0.001; **** P < 0.0001. ( D ) Control and shNRP2-2 BT549 cells were treated with 1400W (50 μM) for 24 hours, and NFE2L2 activation was assessed by its nuclear-to-cytoplasmic ratio based on immunofluorescence ( n = 3). **** P < 0.0001. ( E ) NRP2-depleted BT549 cells were treated with either DMSO or the NO donor SNAP (50 μM) for 24 hours, and NFE2L2 localization was assessed by immunofluorescence ( n = 3). ** P < 0.01; *** P < 0.001. KEAP1 S-nitrosylation was detected by ( F ) biotin switch assay and ( G ) iodoTMT assay in control and NRP2-knocked down cells with immunoprecipitated KEAP1 used as a control. ( H ) Clonogenic assay of BT549 NRP2 knockdown cells engineered to express ca NFE2L2 or empty vector and irradiated (0–6 Gy; n = 2, representative image). * P < 0.05. ( I ) NFE2L2 nuclear/cytoplasmic ratio assessed by IF of control and NRP2-knockdown cells after 4 Gy irradiation every day starting from day 0 until day 5 ( n = 3). *** P < 0.001; **** P < 0.0001. Data are presented as means ± SD ( A – E , H , and I ). Statistical analysis was performed using 2-tailed Student’s t test ( B ), 1-way ANOVA multiple comparisons ( A , C – E , and J ), or 2-way ANOVA multiple comparisons ( H and I ).

Article Snippet: The following antibodies were used for immunoblotting: tubulin (Cell Signaling Technology, 3873), β-actin (Cell Signaling Technology, 3700S), GAPDH (14C10) (Cell Signaling Technology, 2118S), human NRP2 (aNRP2-36v2 obtained from aTyr; ref. ), mouse NRP2 (R&D Systems, AF2215), human NOS2 (Cell Signaling Technology, 39898), mouse NOS2 (D6B6S) (Cell Signaling Technology, 13120), nitrotyrosine antibody (Santa Cruz Biotechnology, sc-32757), Gli1 (Cell Signaling Technology, 2553s), KEAP1 (D6B12) (Cell Signaling Technology, 8047s), and phospho-histone H2A.X (Ser139) (20E3) (Cell Signaling Technology, 9718s).

Techniques: Immunofluorescence, Staining, Control, Knockdown, Expressing, Activation Assay, Biotin Switch Assay, Immunoprecipitation, Clonogenic Assay, Plasmid Preparation, Irradiation

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-2–expressing breast cancer cells mitigate radiation-induced oxidative stress through nitric oxide signaling

doi: 10.1172/JCI181368

Figure Lengend Snippet: ( A ) 4T1 cells (5 × 10 5 ) were injected into the mammary fat pads of BALB/c mice. Once the tumor volume reached approximately 100 mm 3 , the mice were divided into 4 groups of 7 mice each (mouse IgG, 0 Gy; mouse IgG, 10 Gy; aNRP2, 0 Gy; aNRP2, 10 Gy). The mice were given i.p. injections of the specified antibody (25mg/kg) every 48 hours starting 1 day prior to irradiation for 2 weeks. Tumors were extracted on day 18 and were used for histological and molecular profiling. ** P < 0.01. ( B ) Necrotic areas of tissue sections of tumors were measured after H&E staining by finding the fraction of the area that is necrotic compared with the area of the tumor ( n = 4). *** P < 0.001. ( C ) Immunoblot showing γ-H2AX protein levels in irradiated tumors that had been treated with either mIgG or aNRP2-28. ( D ) Cell proliferation in tumors from each treatment group was measured by Ki-67 immunofluorescence and quantified as a percentage of cells that were positive ( n = 4). Scale bars: 100 μm. *** P < 0.001. ( E ) NOS2 mRNA and ( F ) NOS2 protein levels were quantified for each treatment group using qPCR and immunoblotting, respectively ( n = 3). **** P < 0.0001. ( G ) mRNA expression of NFE2L2 target genes ( SLC7A11 and HMOX1 ) was measured for each treatment group using qPCR ( n = 3). ** P < 0.01; *** P < 0.001; **** P < 0.0001. Data are presented as means ± SEM ( A ) and mean ± SD ( B , D , E , and G ). Statistical analysis was performed using 2-tailed Student’s t test ( D ), 1-way ANOVA multiple comparisons ( B , E , and G ), or 2-way ANOVA multiple comparisons ( A ).

Article Snippet: The following antibodies were used for immunoblotting: tubulin (Cell Signaling Technology, 3873), β-actin (Cell Signaling Technology, 3700S), GAPDH (14C10) (Cell Signaling Technology, 2118S), human NRP2 (aNRP2-36v2 obtained from aTyr; ref. ), mouse NRP2 (R&D Systems, AF2215), human NOS2 (Cell Signaling Technology, 39898), mouse NOS2 (D6B6S) (Cell Signaling Technology, 13120), nitrotyrosine antibody (Santa Cruz Biotechnology, sc-32757), Gli1 (Cell Signaling Technology, 2553s), KEAP1 (D6B12) (Cell Signaling Technology, 8047s), and phospho-histone H2A.X (Ser139) (20E3) (Cell Signaling Technology, 9718s).

Techniques: Injection, Irradiation, Staining, Western Blot, Immunofluorescence, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-2–expressing breast cancer cells mitigate radiation-induced oxidative stress through nitric oxide signaling

doi: 10.1172/JCI181368

Figure Lengend Snippet: ( A ) 4T1 cells (5 × 10 5 ) were injected into the mammary fat pads of BALB/c mice. Once the tumor volume reached approximately 100 mm 3 , the mice were divided into 4 groups of 5 mice each (mouse IgG, 2Gyx5; mouse IgG, 2Gyx5; aNRP2, 2Gyx5; aNRP2, 2Gyx5). The mice were given i.p. injections of the specified antibody (25 mg/kg) every 48 hours starting 1 day prior to irradiation for 2 weeks. Tumor volumes were measured with calipers every 2 days and are shown as means ± SEM. Tumors were extracted on day 16 and were used for histological and molecular profiling. ** P < 0.01. ( B ) Necrotic areas of tissue sections of tumors were measured after H&E staining by finding the fraction of the area that is necrotic compared with the area of the tumor ( n = 5).* P < 0.05. ( C ) Immunoblot showing γ-H2AX protein levels in irradiated tumors that had been treated with either mIgG or aNRP2-28. ( D ) NOS2 mRNA and protein levels were quantified for each treatment group using qPCR and immunoblotting ( n = 3). * P < 0.05. ( E ) mRNA expression of NFE2L2 target genes ( SLC7A11 and HMOX1 ) was measured for each treatment group using qPCR ( n = 3). * P < 0.05; ** P < 0.01. Data are presented as means ± SD ( B , D , and E ). Statistical analysis was performed using 2-tailed Student’s t test ( B , D , and E ) or 2-way ANOVA multiple comparisons ( A ).

Article Snippet: The following antibodies were used for immunoblotting: tubulin (Cell Signaling Technology, 3873), β-actin (Cell Signaling Technology, 3700S), GAPDH (14C10) (Cell Signaling Technology, 2118S), human NRP2 (aNRP2-36v2 obtained from aTyr; ref. ), mouse NRP2 (R&D Systems, AF2215), human NOS2 (Cell Signaling Technology, 39898), mouse NOS2 (D6B6S) (Cell Signaling Technology, 13120), nitrotyrosine antibody (Santa Cruz Biotechnology, sc-32757), Gli1 (Cell Signaling Technology, 2553s), KEAP1 (D6B12) (Cell Signaling Technology, 8047s), and phospho-histone H2A.X (Ser139) (20E3) (Cell Signaling Technology, 9718s).

Techniques: Injection, Irradiation, Staining, Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-2–expressing breast cancer cells mitigate radiation-induced oxidative stress through nitric oxide signaling

doi: 10.1172/JCI181368

Figure Lengend Snippet: ( A ) Schematic of the fractionated radiation and antibody-treatment schedules. ( B ) Tumor volumes in mice that had been implanted orthotopically with a human TNBC PDX in NSG mice. The mice were divided into 4 groups of 5 mice each. When tumors reached approximately 125–150 mm 3 , the mice were treated with either IgG (10 mg/kg), aNRP2-10 (10 mg/kg), IgG 8Gyx3, or aNRP2-10 8Gyx3. Antibody treatments were given as i.p. injections. The waterfall plot shows the percentage change in growth of the tumor from day –1 to day 15 for each individual mouse. Molecular and histological analysis of the tumors were done on day 15 after the first radiation dose. *** P < 0.001; **** P < 0.0001. ( C ) The tumor weights from the radiation-treated groups ( n = 5), and percentage of tumor necrosis based on H&E section of the tumors from the radiation-treated groups ( n = 3). ** P < 0.01; *** P < 0.001. ( D ) Immunoblot of γ-H2AX from 3 mice in each of the fractionated radiation-treated groups. ( E ) NOS2 , HMOX1, and PRDX1 mRNA expression was quantified by qPCR from 3 mice in each of the radiation-treated groups. * P < 0.05; *** P < 0.001. Data are presented as means ± SD ( C and E ). Statistical analysis was performed using 2-tailed Student’s t test ( C and E ) or 1-way ANOVA multiple comparisons ( B ).

Article Snippet: The following antibodies were used for immunoblotting: tubulin (Cell Signaling Technology, 3873), β-actin (Cell Signaling Technology, 3700S), GAPDH (14C10) (Cell Signaling Technology, 2118S), human NRP2 (aNRP2-36v2 obtained from aTyr; ref. ), mouse NRP2 (R&D Systems, AF2215), human NOS2 (Cell Signaling Technology, 39898), mouse NOS2 (D6B6S) (Cell Signaling Technology, 13120), nitrotyrosine antibody (Santa Cruz Biotechnology, sc-32757), Gli1 (Cell Signaling Technology, 2553s), KEAP1 (D6B12) (Cell Signaling Technology, 8047s), and phospho-histone H2A.X (Ser139) (20E3) (Cell Signaling Technology, 9718s).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: A novel bioreactor technology for modelling fibrosis in human and rodent precision-cut liver slices

doi: 10.1101/331173

Figure Lengend Snippet: ( A-B ) Graphs show fibronectin (μg/ml) and hyaluronic acid (ng/ml) levels in the media of bioreactor-cultured hPCLS after 24 rest and then 24h or 72h culture ± fib stim (TGFβ1/PDGFββ) ± Alk5i, numbers on bars show fold change compared to 48h control. ( C ) Graphs show mRNA levels of collagen 1A1, αSMA and TIMP1 in hPCLS at t=0 and after 24h rest plus 72h culture ± fib stim ± Alk5i, numbers on bars show fold change compared to t=0. ( D ) Representative 100x magnification images of αSMA (left panel) and Picrosirius red (right panel) stained hPCLS from three different donor livers at t=0, and after 24h rest plus 72h culture (4-days) ± fib stim ± Alk5i. Scale bars equal 200 µm. ( E ) Representative 200x magnification image of Picrosirius red stained hPCLS form donor 4. ( F ) Graph shows the percentage area of picrosirius red stained tissue in hPCLS at t=0, and after 24h rest plus 72h culture ± fib stim ± Alk5i (96h total culture). Data are mean ± SEM in n=4 independent slice experiments. P values were calculated using an Anova with Tukey’s multiple comparisons test (* P <0.05, ** P <0.01, *** P <0.001 and **** P <0.0001).

Article Snippet: ELISA quantifications for rat COL1a1 (LS-F11152, LSBio, UK), human (E88-129) and rat (E110-125) albumin (Bethyl laboratories, UK), human fibronectin (DY1918-05, R&D systems, UK) and human hyaluronic acid (DY2089, R&D systems, UK) were performed as per manufacturer’s instructions.

Techniques: Cell Culture, Staining

Journal: bioRxiv

Article Title: RGG peptide induces the disassembly of disease-relevant FUS and TDP43 condensates

doi: 10.1101/2025.03.19.643735

Figure Lengend Snippet: (A) Graph representing the distribution of FUS-P525L protein in nucleus and cytoplasm. The fluorescent intensities of the nucleus and cytoplasm were calculated from the experiment as performed in , and the ratio is plotted here. A student-paired t-test was used to calculate the significance value (n=6). (B) Incucyte images (real-time cell death analysis) representing the cellular uptake of propidium iodide (PI) in different conditions in HeLa cells. Scale bar=100um. (C) Graph representing the number of propidium iodide (PI) positive cells (dead cells) from the experiment as performed in B. The time on the x-axis reflects the time-point after transfection in hours. The significance was calculated using 2-way ANOVA with multiple comparisons (n=4). Error bars in all graphs represent mean ±SEM, and the same color points in A depict the data from a single experimental set. *, **, ***, and **** denote p-value≤0.05, ≤0.01, ≤0.001, and ≤0.0001, respectively.

Article Snippet: The cell death analysis was performed using the IncuCyte S3 live-cell analysis instrument (Sartorius), and the change in the number of PI-positive cells (dead cells) in different conditions was plotted in the graph.

Techniques: Transfection

Journal: bioRxiv

Article Title: RGG peptide induces the disassembly of disease-relevant FUS and TDP43 condensates

doi: 10.1101/2025.03.19.643735

Figure Lengend Snippet: Incucyte images representing the cellular uptake of propidium iodide (PI) in different conditions in HeLa cells. Scale bar=100um. The images are part of and .

Article Snippet: The cell death analysis was performed using the IncuCyte S3 live-cell analysis instrument (Sartorius), and the change in the number of PI-positive cells (dead cells) in different conditions was plotted in the graph.

Techniques:

Journal: bioRxiv

Article Title: YAP1 status defines two intrinsic subtypes of LCNEC with distinct molecular features and therapeutic vulnerabilities

doi: 10.1101/2023.12.19.572449

Figure Lengend Snippet: YAP1 status does not predict sensitivity to cisplatin in LCNEC cell lines (A) or cell line/PDX xenograft models (B). C, Treatment of LCNEC cell lines with DMSO, cisplatin, MYF-01-37, XAV-939 and decitabine did not change YAP1, but verteporfin reduced YAP1 levels. D, YAP1 status does not predict response to verteporfin. E, Waterfall plot of drug sensitivity in LCNEC cell lines. A comparison of high YAP1 levels and IC50 values identified several drugs with similar targets, including MEK1/2, CDK4/6, and Src family kinase inhibitors. F, Treatment of YAP1-high LCNEC and SCLC (SW1271) cell lines with trametinib did not change YAP1 levels. G, Tumor growth curves from YAP1-high cell line xenografts H1915 (LCNEC) and SW1271 (SCLC) demonstrate a delay in tumor growth with trametinib treatment.

Article Snippet: One million cells each for H810, H1155, H1755, H1833, H2106, MKL1, HCC2374, HCC4017, HOP92, H1359, H2066, H1770, H2106, H1570, H661, HCC3051, H460, HCC4017, H1299, and H1915 in triplicate were surfaced stained with DLL3 (FAB4315P; R&D Systems), AXL (386202; Biolegends), CD56 (362534), EPCAM (324222) or IgG control and then fixed in 2% PFA.

Techniques: Comparison

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