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: Scientific Reports

Article Title: Unveiling novel insights into human IL-6 − IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis

doi: 10.1038/s41598-024-69429-w

Figure Lengend Snippet: Affinity kinetics between hIL-6 mutants and hIL-6R − gp130 assayed by SPR. gp130 was immobilized onto a CM5 chips via standard amine coupling and different concentrations of hIL-6 mutants with a saturated concentration of hIL-6R were pre incubated and the mixture were injected. Affinity kinetics were analyzed by the Langmuir’s 1:1 model fit on seven serial dilutions of hIL-6 mutants from 250 nM and flow speed of 30 μL/min. ( A ) Schematic diagram of binding affinity between hIL-6 mutants and hIL-6R − gp130 by SPR; ( B – K ) representative examples of curve fits for the affinity kinetic analysis of hIL-6 mutants.

Article Snippet: The assay was optimized according to the description provided in reference . monomeric gp130 (Sino Biological, Beijing, China, Cat. 10974-HCCH1) was immobilized on CM5 sensor chips (GE Healthcare) by standard amine coupling method, as recommended by the manufacturer.

Techniques: Concentration Assay, Incubation, Injection, Binding Assay

Journal: Scientific Reports

Article Title: Unveiling novel insights into human IL-6 − IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis

doi: 10.1038/s41598-024-69429-w

Figure Lengend Snippet: Affinity of the interaction between hIL-6/mutants and hIL-6R/gp130 by Biacore SPR.

Article Snippet: The assay was optimized according to the description provided in reference . monomeric gp130 (Sino Biological, Beijing, China, Cat. 10974-HCCH1) was immobilized on CM5 sensor chips (GE Healthcare) by standard amine coupling method, as recommended by the manufacturer.

Techniques: Mutagenesis, Binding Assay

Journal: Scientific Reports

Article Title: Unveiling novel insights into human IL-6 − IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis

doi: 10.1038/s41598-024-69429-w

Figure Lengend Snippet: The spatial arrangement of amino acid residues hIL-6 R167 and E171 in the hexameric hIL-6 − hIL-6R − gp130 complex (PDB code: 1p9m). ( A ) Close spatial proximity is observed between hIL-6 R167 and E171 and the hIL-6 AB-loop; ( B ) the potential influence of hIL-6 R167 on the interaction between hIL-6 K53 and hIL-6R Q95 is indicated. The hIL-6 molecule is shown in brown, hIL-6R in purple, and gp130 in green.

Article Snippet: The assay was optimized according to the description provided in reference . monomeric gp130 (Sino Biological, Beijing, China, Cat. 10974-HCCH1) was immobilized on CM5 sensor chips (GE Healthcare) by standard amine coupling method, as recommended by the manufacturer.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: TP 53 INP 1 inhibits hypoxia‐induced vasculogenic mimicry formation via the ROS /snail signalling axis in breast cancer

doi: 10.1111/jcmm.13625

Figure Lengend Snippet: Relationship between TP53INP1 expression and clinicopathologic features, VM formation in breast cancer

Article Snippet: MCF‐7 cells were transfected with TP53INP1 shRNA (HSH022738‐LvRU6MP) or shRNA control (CSHCTR001‐LVRU6MP) purchased from GeneCopoeia, Inc.

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: TP 53 INP 1 inhibits hypoxia‐induced vasculogenic mimicry formation via the ROS /snail signalling axis in breast cancer

doi: 10.1111/jcmm.13625

Figure Lengend Snippet: TP53INP1 expression in breast cancer tissues and pericarcinous tissues

Article Snippet: MCF‐7 cells were transfected with TP53INP1 shRNA (HSH022738‐LvRU6MP) or shRNA control (CSHCTR001‐LVRU6MP) purchased from GeneCopoeia, Inc.

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: TP 53 INP 1 inhibits hypoxia‐induced vasculogenic mimicry formation via the ROS /snail signalling axis in breast cancer

doi: 10.1111/jcmm.13625

Figure Lengend Snippet: Difference of VE‐cadherin, Snail and HIF‐1α expression in TP53INP1‐positive and TP53INP1‐negative groups

Article Snippet: MCF‐7 cells were transfected with TP53INP1 shRNA (HSH022738‐LvRU6MP) or shRNA control (CSHCTR001‐LVRU6MP) purchased from GeneCopoeia, Inc.

Techniques: Expressing

Journal: Diabetologia

Article Title: Human adipose microRNA-221 is upregulated in obesity and affects fat metabolism downstream of leptin and TNF-α

doi: 10.1007/s00125-013-2950-9

Figure Lengend Snippet: miR-221 targets the 3′ UTRs of ADIPOR1 and ETS1 and decreases their protein levels. ( a,b ) TargetScan conserved predicted miR-221 binding site on the 3′ UTRs of ADIPOR1 and ETS1 . ( c,d ) Quantification of dual-luciferase assay in HEK 293 cells co-transfected with miR-221 mimic (black bars) or control oligonucleotide (white bars) and ADIPOR1/2 ( c ) or ETS1 ( d ) 3′ UTR reporter plasmids. ( d ) Two overlapping fragments of the ETS1 3′-UTR were subcloned into separate vectors, indicated (A) and (B). Firefly/renilla luciferase ratio (luc ratio) was normalised to no-UTR controls. Error bars indicate SD ( n = 5). * p < 0.05 (two-tailed Mann–Whitney U test). ( e ) Quantification of QRT-PCR for ETS1 mRNA in human pre-adipocytes transfected with miR-221 mimic (black bars) or control oligonucleotide (white bars). Error bars indicate SD ( n = 3). ( f ) Immunoblots for ADIPOR1, ETS1 and β-actin in human pre-adipocytes transfected with miR-221 mimic (+) or control oligonucleotide. ( g ) Quantification of the immunoblots shown in ( f ). Background-subtracted mean signal ( n = 2) for ADIPOR1 and ETS1, normalised to the loading control (β-actin). Black bars, miR-221 mimic; white bars, control oligonucleotide

Article Snippet: For 3′ untranslated (UTR) reporter assays, JetPrime (Polyplus-transfection SA, Illkirch, France) was used to co-transfect HEK 293 cells (ATCC) with miRNA 3′ UTR target expression vectors for human ETS1 , ADIPOR1 , ADIPOR2 or control (Genecopoeia, Germantown, MD, USA), and human miR-221 mimic (Dharmacon, Lafayette, CO, USA) or scrambled control oligonucleotide, in 24-well plates.

Techniques: Binding Assay, Luciferase, Transfection, Control, Two Tailed Test, MANN-WHITNEY, Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: Delta/Notch-Like EGF-Related Receptor (DNER) Is Not a Notch Ligand

doi: 10.1371/journal.pone.0161157

Figure Lengend Snippet: (A) Pooled luciferase results from 4 separate experiments (normalized to the mean of empty vector in each experiment). U2OS cells were transfected with ligand (DLL1, DNER, or EV), and separately a population of U2OS cells was transfected to express Notch, the control luciferase Renilla , and TP1, a promoter that expresses firefly luciferase when Notch is activated. The two populations were co-cultured 24 hours after transfection ( trans configuration), and activity read after an additional 24–48 hours of incubation. (B) C2C12 cells (myoblasts) were incubated with differentiation media (2% horse serum) that either had pre-clustered DLL1-fc (1:1), pre-clustered DNER-fc (1:1), un-clustered DNER-fc, or fc only, all at a ratio of 1:150 in media. Cells were incubated for 72 hours, then fixed, and stained for the presence of myosin heavy chain (MHC) and nuclei. By measuring the percent of total nuclei that were inside of differentiated MHC positive myotubes, fusion indexes were calculated. (C) DNER (top left, green) transfected U2OS cells were not labeled by pre-clustered Notch-fc (top middle, red) but DLL1 (bottom left, green) transfected U2OS cells were labeled by pre-clustered Notch-fc (bottom middle, red). Merged images are shown at far right. Scale 10 μM. **** = p value <0.0001. *** = p value 0.002. ns = not significant. DLL1 = Delta-like 1, a known Notch Ligand, DNER = Delta/Notch-like epidermal growth factor (EGF) related receptor, GSI = γ-secretase inhibitor, fc only = rabbit anti-human-fc.

Article Snippet: Transient transfection of cultured cells was accomplished with the Fugene/Optimem (Promega) system and according to the manufacturer’s instructions with the following plasmids at a concentration, unless otherwise stated, of 0.1 μg/well of DNA per plasmid of a 96-well plate: DNER (generous gift of Drs. De Graaff and Sillevis-Smitt, Erasmus Medical Center, Rotterdam the Netherlands, originally from M. Kengaku), DLL1 (EX-Y3540-M11, GeneCopoeia, Rockville, MD, USA), empty vector on a p-receiver backbone (GeneCopoeia, Rockville, MD, USA), Renilla (luciferase control, 0.01 μg/well, pRL-TK, Promega E2241), TP1 (very sensitive intracellular Notch reporter with twelve CSL binding sites converting intracellular Notch activity to firefly luminescence, which is capable of detecting slight perturbations in signaling strength with little background), and full length human Notch1 on a MigR1 backbone (both generous gifts of Dr. Jon Aster).

Techniques: Luciferase, Plasmid Preparation, Transfection, Control, Cell Culture, Activity Assay, Incubation, Staining, Labeling

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Photograph of the cut section of an ovarian sclerosing stromal tumor (SST; left) displaying classic SST appearance with yellow tissue at periphery and white, central fibrotic depression, and micrographs of hematoxylin & eosin stained representative section at low (top right) and high (bottom right) magnification. Scale bars, 1 cm (left), 200 μm (top right), 50 μm (bottom right). b Schematic representation of the FHL2-GLI2 fusion transcript including the exons and domains involved. The breakpoint of the 5′ and 3′ partner genes are represented as black vertical lines. Spanning reads are depicted and aligned to the predicted junction sequence. c Schematic representation showing the Reads Per Kilobase per Million (RPKM) mapped read counts of each GLI2 exon. The GLI2 fusion breakpoint is represented as a red dashed line. d Fluorescence in situ hybridization (FISH) of two representative SSTs using a three-color FHL2-GLI2 probe, with 5′ GLI2 (orange), 3′ GLI2 (red), and 5′ FHL2 (green), showing the presence of the FHL2-GLI2 fusion (white arrows). e Representative Sanger sequencing electropherograms of the genomic FHL2-GLI2 breakpoint. f RNA in situ hybridization (RNA-ISH) using custom FHL2-GLI2 probes (red) showing the chimeric FHL2-GLI2 mRNA expression in two representative SSTs harboring the FHL2-GLI2 fusion. g Frequency of the FHL2-GLI2 fusion gene and GLI2 rearrangements in 26 SSTs from this study. h Frequency of the FHL2-GLI2 fusion gene and GLI2 rearrangements in 26 SSTs and frequency of the FHL2-GLI2 fusion gene in 48 other ovarian sex cord-stromal tumors from this study. aGCT, adult-type granulosa cell tumor. i Frequency of FHL2-GLI2 fusion gene in 26 SSTs from this study and in 9950 tumors from 33 cancer types from The Cancer Genome Atlas (TCGA). AML acute myeloid leukemia, PCPG pheochromocytoma and paraganglioma.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Staining, Sequencing, Fluorescence, In Situ Hybridization, RNA In Situ Hybridization, Expressing

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Cell titer blue proliferation assay of immortalized mesenchymal stem cells (MSCs), HEK-293, medulloblastoma (DAOY), and human basal cell carcinoma (BCC) cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or the FHL2-GLI2 fusion. b Representative images of colony formation assay of MSCs, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 (scale bars, 5 mm; top). Quantification of the number of colonies/well compared to control (bottom). c Wound healing assay of MSCs, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2 or FHL2-GLI2. The migratory effects/wound area was assessed at 0 and 24 h (Scale bar, 500 μm; top) and quantified (bottom). In a – c , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Proliferation Assay, Stable Transfection, Expressing, Plasmid Preparation, Control, Colony Assay, Wound Healing Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Quantitative assessment of CALB2 transcripts in immortalized mesenchymal stem cells (MSCs) and HEK-293 cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or the FHL2-GLI2 fusion. Expression levels were normalized to GAPDH expression, and comparisons of mRNA expression levels were performed relative to control. b Representative western blot analysis of calretinin protein levels in MSCs and HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Tubulin was used as protein loading control. Quantification (bottom) of protein levels as compared to control. c Representative confocal micrographs of immunofluorescence analysis of calretinin (green) and 4–6-diamidino-2-phenylindole (DAPI, blue) in MSCs and HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 (scale bars, 50 μm). Quantification (bottom) of calretinin intensity/cell relative to control. d Quantitative assessment of FOXL2 transcripts in MSCs and HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Expression levels were normalized to GAPDH expression, and comparisons of mRNA expression levels were performed relative to control. In a – d , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Control, Western Blot, Immunofluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Differential gene expression analysis of human sclerosing stromal tumors (SSTs) subjected to RNA-sequencing ( n = 8, this study) and high-grade serous ovarian carcinomas ( n = 16; The Cancer Genome Atlas) and other sex cord-stromal tumors (SCSTs, n = 11; this study). Gene expression fold-change is color-coded according to the legend. Only genes significantly differentially expressed ( P < 0.05; two-tailed unpaired t -test) are shown. CPM, count per million. b Expression levels of Sonic Hedgehog (SHH) pathway genes in human SSTs ( n = 11) and other sex-cord stromal tumors ( n = 9) as defined using NanoString. Expression levels and SHH enrichment scores are color-coded according to the legends. *** P < 0.001, Wilcoxon rank test. Hierarchical clustering was performed using complete linkage and Euclidian distance. c Quantitative assessment of the Sonic Hedgehog pathway PTCH1 and GLI1 transcripts in immortalized mesenchymal stem cells (MSCs), HEK-293 and medulloblastoma (DAOY) cells and of PTCH1 and CCND1 transcripts in human basal cell carcinoma (BCC) cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or FHL2-GLI2. Expression levels were normalized to GAPDH expression, and comparisons of mRNA expression levels were performed relative to control. d Representative western blot analysis of PTCH1 and GLI1 protein expression in MSC, HEK-293 and DAOY cells and of PTCH1 and CCND1 in BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Tubulin was used as protein loading control. Quantification (below) of protein levels as compared to control. In c – d , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Gene Expression, RNA Sequencing, Two Tailed Test, Expressing, Stable Transfection, Plasmid Preparation, Control, Western Blot

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Representative confocal micrographs of immunofluorescence analysis of FLAG (red), 4–6-diamidino-2-phenylindole (DAPI, blue), and GFP (green) in HEK-293 cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or FHL2-GLI2. Scale bars, 10 μm. b GLI response element (GLI-RE) luciferase reporter assay of HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 (top), and GLI-RE promoter activity in HEK-293 transiently transfected with control, FHL2-GLI2 and FHL2-GLI2 with R338A-K339A mutations in the Zinc Finger 5 of GLI2 required for DNA binding (FHL2-GLI2 Mutated; bottom). SV40-Renilla was used to normalize transfection efficiency. c Immunoprecipitation assay with SUFU antibody of HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Western blot analysis using anti-FLAG and anti-SUFU antibodies, and tubulin as loading control (left). GLI-RE promoter activity in HEK-293 stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 transfected with SUFU or control (right). d Cell titer blue proliferation assay of HEK-293, DAOY and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 treated with 20 µM GANT61 or vehicle control (DMSO). GANT, GANT61. e FLAG Chromatin Immunoprecipitation (ChIP) assay of GLI1 and PTCH1 promoters (promoter 1 and 2) in MSC and HEK-293 cells stably expressing either control or FHL2-GLI2. GLI1 and PTCH1 gene body and MYOD1, gene promoters not under GLI regulation, were used as negative controls. In b – d , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; *P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Reporter Assay, Activity Assay, Transfection, Binding Assay, Immunoprecipitation, Western Blot, Proliferation Assay, Chromatin Immunoprecipitation, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Cell titer blue proliferation assay of immortalized mesenchymal stem cells (MSCs), HEK-293, medulloblastoma (DAOY) and human basal cell carcinoma (BCC) cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2) or FHL2-GLI2 treated with 250 nM Vismodegib or vehicle control (DMSO). b Representative images of colony formation assay of MSC, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 treated with Vismodegib 500 nM or vehicle control (DMSO). Scale bars, 5 mm. Quantification of the number of colonies/well compared to control (bottom). c Wound healing assay of MSC, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 treated with 250 nM Vismodegib or vehicle control (DMSO). The migratory effect/wound area was assessed at 0 and 24 h and quantified compared to DMSO (bottom). Vismo, Vismodegib. Scale bars, 500 μm. In a – c , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: Human wild-type FHL2 (EX-M0686-Lv102), wild-type GLI2 (EX-Y4001-Lv102), truncated GLI2 lacking the N-terminal domain constructed from GLI2 (EX-Y4001-Lv102), FHL2-GLI2 (CS-Y4001-Lv102–01) ORF cDNA clones with N-terminal FLAG tag were purchased from GeneCopoeia.

Techniques: Proliferation Assay, Stable Transfection, Expressing, Plasmid Preparation, Control, Colony Assay, Wound Healing Assay, Two Tailed Test