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 BT-549, BT-20, MDA-MB-468, and Hs578t human breast cancer cell lines and the 4T1 mouse breast cancer cell line were purchased from ATCC and were authenticated by the University of Arizona Genetic Core (UAGC).

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 ) 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 BT-549, BT-20, MDA-MB-468, and Hs578t human breast cancer cell lines and the 4T1 mouse breast cancer cell line were purchased from ATCC and were authenticated by the University of Arizona Genetic Core (UAGC).

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 BT-549, BT-20, MDA-MB-468, and Hs578t human breast cancer cell lines and the 4T1 mouse breast cancer cell line were purchased from ATCC and were authenticated by the University of Arizona Genetic Core (UAGC).

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 ) 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 BT-549, BT-20, MDA-MB-468, and Hs578t human breast cancer cell lines and the 4T1 mouse breast cancer cell line were purchased from ATCC and were authenticated by the University of Arizona Genetic Core (UAGC).

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 BT-549, BT-20, MDA-MB-468, and Hs578t human breast cancer cell lines and the 4T1 mouse breast cancer cell line were purchased from ATCC and were authenticated by the University of Arizona Genetic Core (UAGC).

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