Journal: Physiological Reports

Article Title: Influence of bacterial components on the developmental programming of enteric neurons

doi: 10.14814/phy2.14611

Figure Lengend Snippet: Characterization of ENCDC cultures. Flow cytometry of ENCDC cultures stained with antibodies against the immature neuronal marker, p75 NTR+ ‐FITC demonstrated 95.1% p75 NTR+ ‐positive, 97.3% viability in subculture 3, and 96.1% p75 NTR+ ‐positive, 98.2% viability in subculture 4 (a). Proportion of ENCDC cultures characterized for expression of p75NTR+, pH3, 5‐HT, nNOS, and TH. Scale bar = 20 µm (b). There was a significant increase in the proportion of serotonergic neurons and nitrergic neurons in subculture 5 (gray) compared to subculture 4 (black), but no significant difference in proliferating cells or dopaminergic neurons across the cultures. *p ≤ .05. Values are presented as mean ± SEM (c)

Article Snippet: Single cells were incubated with primary antibody rabbit anti‐mouse p75 NTR+ for 1 hr (1:50, Alomone ANT‐007), and secondary antibody anti‐rabbit IgG microbeads for 15 min (150 µl per 10 7 cells; MACS Miltenyi Biotec 130‐048‐602).

Techniques: Flow Cytometry, Staining, Marker, Expressing

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Design considerations for single and dual-receptor engineered T cells (A) Schematic representation of a DAP12-associated synthetic antigen receptor (DAP12-SAR) composed of an antigen binding domain fused to the hinge, transmembrane (TM) and intracellular (ICD) domains of a DAP12-associated activating receptor (created using BioRender). (B) Schematic diagram of cDNA encoding DAP12 and the SAR separated by a Thoseasigna virus 2A (T2A) sequence for co-expression. (C) SAR surface expression was determined by binding of a myc-tag specific mAb or HER2-Fc to T cells engineered with the IL13Rα2-KIR and HER2-KIR, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + . Presented is SAR expression on the CD4 + NGFR + population (unfilled black = non-transduced; gray = SAR). (D) Representative cytotoxicity of IL13Rα2-KIR, HER2-KIR and non-specific SAR T cell products after 96-h co-culture with U-251 tumor cells in an Incucyte assay (E:T = 8:1). The experiment was performed in technical triplicates and error bars are standard error mean (SEM). (E) Schematic representation of dual-SAR constructs. (F) SAR surface expression on T cells engineered with single and dual SAR constructs, using anti-Myc tag mAb and HER2-Fc to detect binding of the IL13Rα2-KIR and HER2-KIR, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + .

Article Snippet: All proliferation assay samples were incubated for 3 days at 37°C and stained with Live/Dead Fixable Near-IR stain (Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (eBioscience) and VioBright FITC-conjugated mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Binding Assay, Virus, Sequencing, Expressing, Co-Culture Assay, Construct

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Dual targeting of IL13Rα2 and HER2 can be achieved by expression of different synthetic DAP12-associated receptors (A) Schematic representation of various HER2-SAR constructs evaluated. (B) Receptor surface expression and transduction efficiency of various HER2-SAR constructs, determined by binding to HER2-Fc and tNGFR expression, respectively. (C) Cytotoxicity of HER2-SAR engineered T cells after 120 h co-culture with HCT-116 and U-251 tumor cells in an Incucyte assay. (D) Proliferation of respective HER2-SAR constructs after 72 h co-culture with HCT-116 or U-251 tumor cells at a 1:1 ratio. Absolute cell count was determined by flow cytometry using 123count eBeads. (E) Schematic diagram of cDNA encoding single and dual SAR constructs. (F) SAR surface expression of single and dual IL13Rα2/HER2 SAR T cells determined by binding of a myc-tag specific mAb or HER2-Fc to detect the IL13Rα2-KIR and HER2-TREM1 SARs, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + . Presented is SAR expression on the CD4 + NGFR + population. (G) Mean fluorescence intensity (MFI) of respective SARs on single and dual-receptor engineered T cells. Data are from 4 experiments with 4 PBMC donors (each donor is represented by a unique symbol). (H) Cytotoxicity of single, dual, and non-transduced T cell products after 120-h co-culture with U-251 tumor cells in an Incucyte assay. All conditions were normalized to growth of tumor cells alone. Data are from 4 individual experiments with 3 PBMC donors. Error bars represent SEM of technical replicates. (I) Cytotoxicity of single, dual, and non-transduced T cell products after 120-h co-culture with U-251 IL13Rα2 KO and U-251 HER2 KO cells in an IncuCyte assay. Data are representative of 3 experiments with 3 PBMC donors. Error bars represent SEM. Statistical analysis for (G) and (H) were performed using a paired t test and two-way ANOVA with correction for multiple comparison (Tukey test), respectively (∗ p = ≤ 0.05, ∗∗ p = ≤ 0.01, ∗∗∗ p = ≤ 0.001, ∗∗∗∗ p = ≤ 0.0001).

Article Snippet: All proliferation assay samples were incubated for 3 days at 37°C and stained with Live/Dead Fixable Near-IR stain (Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (eBioscience) and VioBright FITC-conjugated mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Expressing, Construct, Transduction, Binding Assay, Co-Culture Assay, Cell Counting, Flow Cytometry, Fluorescence, Comparison

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Combinatorial targeting of CD133 and HER2 can be achieved using the dual-SAR approach (A) Schematic diagram of cDNA encoding CD133-SAR constructs. (B) SAR surface expression on primary human T cells as determined by binding of a myc-tag specific mAb or HER2-Fc to detect the IL13Rα2-KIR and HER2-TREM1 SARs, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + . (C) T cells were incubated with firefly luciferase-expressing HCT-116 tumor cells for 20 h at indicated effector:target ratios. Luminescence was read with an open filter upon addition of 0.15 mg/mL D-luciferin substrate and converted to % cytotoxicity. Error bars display standard deviation for technical replicates. (D) T cells were CTV-labeled and incubated for 72-h with HCT-116 tumor cells at a 1:1 ratio. T cell proliferation was measured by flow cytometry with live > CD3 + > CD4 + > NGFR+ cells presented. (E) Schematic diagram of cDNA encoding single and dual CD133/HER2 SAR constructs. (F) SAR surface expression of single and dual CD133/HER2 SAR T cells determined by binding of a FLAG tag specific mAb or HER2-Fc to detect the CD133-NKp44 and HER2-TREM1 SARs, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + . Presented is SAR expression on the CD4 + NGFR + population. (G) Mean fluorescence intensity (MFI) of respective SARs on single and dual-receptor engineered T cells. Data are from 4 experiments with 4 PBMC donors (each donor is represented by a unique symbol). (H) Cytotoxicity of single, dual, and non-transduced T cell products after 72-h co-culture with HCT-116 tumor cells in an Incucyte assay. All conditions in were normalized to growth of tumor cells alone. Data are from 4 individual experiments with 3 PBMC donors. Error bars represent SEM. (I) Cytotoxicity of single, dual and non-transduced T cell products after 120-h co-culture with HCT-116 CD133 KO and HCT-116 HER2 KO cells in an IncuCyte assay. Data are representative of 3 experiments with 3 PBMC donors. Error bars represent SEM of technical replicates. Statistical analysis for (G) and (H) were performed using a paired t test and two-way ANOVA with correction for multiple comparison (Tukey test), respectively (∗ p = ≤ 0.05, ∗∗ p = ≤ 0.01, ∗∗∗ p = ≤ 0.001, ∗∗∗∗ p = ≤ 0.0001).

Article Snippet: All proliferation assay samples were incubated for 3 days at 37°C and stained with Live/Dead Fixable Near-IR stain (Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (eBioscience) and VioBright FITC-conjugated mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Construct, Expressing, Binding Assay, Incubation, Luciferase, Standard Deviation, Labeling, Flow Cytometry, FLAG-tag, Fluorescence, Co-Culture Assay, Comparison

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Expression of multiple receptors attenuates the production of inflammatory cytokines but not proliferation (A and D) Schematic representation of stimulation conditions for single- vs. dual-SAR engagement against (A) U-251 tumor cells or (D) HCT-116 tumor cells. (B and E) Intracellular cytokine production by (B) U-251 or (E) HCT-116 stimulated engineered T cells was measured by flow cytometry. Data are presented as percent NGFR+ CD4 and CD8 T cells producing respective cytokines. Data are from four independent experiments with 3 PBMC donors. Each donor is represented by a unique symbol. For the gating strategy see Figure S6 A. (C and F) Engineered T cells were labeled with CellTrace Violet (CTV) and stimulated with (C) U-251 or (F) HCT-116 tumor cells at a 1:1 ratio for 72 h. T cell proliferation and absolute cell count using 123count eBeads were measured by flow cytometry. Data are from three independent experiments with 2 T cell donors. Each donor is represented by a unique symbol. For the gating strategy see Figure S6 . Statistical analysis for (B), (C), (E), and (F) were performed using two-way ANOVA with correction for multiple comparison (Tukey test) (∗ p = ≤ 0.05, ∗∗ p = ≤ 0.01, ∗∗∗ p = ≤ 0.001, ∗∗∗∗ p = ≤ 0.0001, ns = not significant.

Article Snippet: All proliferation assay samples were incubated for 3 days at 37°C and stained with Live/Dead Fixable Near-IR stain (Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (eBioscience) and VioBright FITC-conjugated mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Expressing, Flow Cytometry, Labeling, Cell Counting, Comparison

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Expression of multiple receptors attenuates the early signal strength of individual receptors (A and B) Intracellular phospho-specific staining of ERK. Cells were stimulated for 30 min with respective WT or KO tumor lines, fixed, methanol-permeabilized and stained for phosphorylated ERK 1/2 (pT202/pY204). (A) Representative plots from 1 of 4 independent experiments and (B) %pERK+ of single and dual-SAR T cells following stimulation with a single target antigen. Data are from 4 independent experiments and 3 T cell donors. (C) Cells were stimulated with HCT-116 CD133 KO or U251 IL13Rα2 KO tumor cells for 1–4 h. The cells were surface stained for viability, CD4, CD8, NGFR and CD69, fixed/permeabilized, and then stained intracellularly/intranuclearly for Nur77. Cells were gated as follows: lymphocytes > single cells > live > CD4/CD8 > NGFR + > Nur77/CD69. Presented is Nur77 and CD69 expression on the CD8 + NGFR + population. Data are generated with T cells from one donor. For the gating strategy, see Figure S6 C.

Article Snippet: All proliferation assay samples were incubated for 3 days at 37°C and stained with Live/Dead Fixable Near-IR stain (Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (eBioscience) and VioBright FITC-conjugated mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Expressing, Staining, Generated

Journal: Bone Reports

Article Title: p75 NTR regulates postnatal skeletal development via NGF-responsive JNK signaling

doi: 10.1016/j.bonr.2025.101854

Figure Lengend Snippet: Global p75 NTR -deficient mice exhibited an osteoporotic phenotype. (A) Photos of 4 and 12-week-old p75 NTR+/+ and p75 NTR−/− mice. Scale bar: 4.0 cm. (B) Body weight curve of p75 NTR+/+ and p75 NTR−/− mice from P0 - P28. Three-dimensional Micro-CT images of 4 (C) and 12 (E) weeks old p75 NTR+/+ and p75 NTR−/− mice's whole femurs, trabecular bones, and cortical bones. The BV/TV, Tb. Th, Tb.N, CtV/TV, Ct. Th and length of femurs of 4 (D) and 12 (F) weeks old p75 NTR+/+ and p75 NTR− /− mice were quantified by 3D Micro-CT. Scale bar (left panel): 1 mm, Scale bar (right panel): 0.5 mm. For 4-week-old mice, n = 5 for each group. For 12-week-old mice, p75 NTR+/+ ( n = 3) and p75 NTR−/− ( n = 4). Alcian blue staining images of the growth plate of 4 (G) and 12 (I) weeks old p75 NTR+/+ and p75 NTR−/− mice's tibias. The growth plate thickness (GP. Th) of 4 (H) and 12 (J) weeks old p75 NTR+/+ and p75 NTR− /− mice were quantified. For 4-week-old mice, n = 5 for each group. For 12-week-old mice, p75 NTR+/+ ( n = 6) and p75 NTR−/− (n = 5). Unpaired Student's t -test was used for analysis, with * p < 0.05, ** p < 0.01, *** p < 0.001. n represents the number of mice analyzed.

Article Snippet: p75 NTR−/− (Stock No: 002213), p75 NTR flox (p75 NTRf/f ) (Stock No: 031162), and paired related homeobox-Cre (Prx1-Cre) (Stock No: 005584) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and housed in the animal facility at the University of California, San Francisco (UCSF) according to institutional guidelines.

Techniques: Micro-CT, Staining

Journal: Bone Reports

Article Title: p75 NTR regulates postnatal skeletal development via NGF-responsive JNK signaling

doi: 10.1016/j.bonr.2025.101854

Figure Lengend Snippet: MSC-specific p75 NTR -deficient mice exhibited an osteoporotic phenotype. (A) PCR results of p75 NTR in p75 NTRf/f and Prx1-Cre; p75 NTR f/f spleen, heart, liver, kidney, muscle, and bone. (B) PCR results of p75 NTR in p75 NTRf/f and Prx1-Cre; p75 NTR f/f monocyte and BMSC. p75 NTR expression was not detected in BMSCs. (C) Photos of 4 and 12-week-old p75 NTRf/f and Prx1-Cre; p75 NTR f/f mice. Scale bar: 4 cm. (D) Body weight of 4 and 12-week-old p75 NTRf/f and Prx1-Cre; p75 NTR f/f mice. For 4-week-old mice, p75 NTRf/f ( n = 3) and Prx1-Cre; p75 NTR f/f ( n = 4). For 12-week-old mice, p75 NTRf/f (n = 5) and Prx1-Cre; p75 NTR f/f (n = 4). Three-dimensional Micro-CT images of 4 (E) and 12 (G) weeks old p75 NTRf/f and Prx1-Cre; p75 NTR f/f mice whole femurs, trabecular bones, and cortical bones. The BV/TV, Tb. Th, Tb.N, CtV/TV, Ct. Th, and length of femurs of 4 (F) and 12 (H) weeks old p75 NTRf/f and Prx1-Cre; p75 NTR f/f mice were quantified by 3D Micro-CT. Scale bar (left panel): 1 mm, Scale bar (right panel): 0.5 mm. For 4-week-old mice, p75 NTRf/f ( n = 4) and Prx1-Cre; p75 NTR f/f ( n = 7). For 12-week-old mice, p75 NTRf/f (n = 6) and Prx1-Cre; p75 NTR f/f (n = 6). Alcian blue staining images of the growth plate of 4 (I) and 12 (K) weeks old p75 NTRf/f and Prx1-Cre; p75 NTR f/f mice's Tibias. The growth plate thickness (GP. Th) of 4 (J) and 12 (L) weeks old p75 NTRf/f and Prx1-Cre; p75 NTR f/f mice were quantified. For both ages, n = 5 for each group. Unpaired Student's t -test was used for analysis, with * p < 0.05, ** p < 0.01, *** p < 0.001. n represents the number of mice analyzed.

Article Snippet: p75 NTR−/− (Stock No: 002213), p75 NTR flox (p75 NTRf/f ) (Stock No: 031162), and paired related homeobox-Cre (Prx1-Cre) (Stock No: 005584) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and housed in the animal facility at the University of California, San Francisco (UCSF) according to institutional guidelines.

Techniques: Expressing, Micro-CT, Staining

Journal: Bone Reports

Article Title: p75 NTR regulates postnatal skeletal development via NGF-responsive JNK signaling

doi: 10.1016/j.bonr.2025.101854

Figure Lengend Snippet: p75 NTR mediates osteogenic differentiation of MSCs. (A) Schematic representation of the osteogenic differentiation process of BMSCs derived from p75 NTR+/+ and p75 NTR−/− mice. Created in BioRender. Kadota, C. (2025) https://BioRender.com/a77c926 (B) Alizarin Red Staining (ARS) images of BMSCs harvested from p75 NTR+/+ and p75 NTR−/− mice cultured in GM and OIM for 21 days, along with quantification of Alizarin Red-positive areas. n = 4 for each group. (C) qRT-PCR analysis of osteoblast differentiation markers in BMSCs cultured in OIM. The expression of osteoblastic differentiation markers, including PTHrP , Runx2 , Dlx5 , Osx , and Alp , were compared, and p75 NTR−/− showed significant downregulation compared to p75 NTR+/+ . n = 3 for each group. Unpaired Student's t -test was used for analysis, with * p < 0.05, ** p < 0.01, *** p < 0.001. n represents the number of experimental replicates.

Article Snippet: p75 NTR−/− (Stock No: 002213), p75 NTR flox (p75 NTRf/f ) (Stock No: 031162), and paired related homeobox-Cre (Prx1-Cre) (Stock No: 005584) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and housed in the animal facility at the University of California, San Francisco (UCSF) according to institutional guidelines.

Techniques: Derivative Assay, Staining, Cell Culture, Quantitative RT-PCR, Expressing

Journal: Bone Reports

Article Title: p75 NTR regulates postnatal skeletal development via NGF-responsive JNK signaling

doi: 10.1016/j.bonr.2025.101854

Figure Lengend Snippet: p75 NTR -deficient BMSCs showed reduced NGF signaling and MAPK activation. (A) Volcano plot shows gene expression changes in bone marrow stem cells (BMSCs) derived from 4-week-old p75 NTR+/+ and p75 NTR−/− mice (n = 3 per group). Expression was profiled using the NanoString nCounter platform in a panel of 477 genes. The x-axis represents the log 2 fold change in p75 NTR−/− relative to p75 NTR+/+ and the y-axis is the –log 10 ( p -value). The dashed horizontal line indicates p = 0.05. Among the 637 genes tested, 15 were significantly downregulated (p < 0.05) in p75 NTR−/− BMSCs. Red dots indicate upregulated genes, while blue dots indicate downregulated genes in p75 NTR−/− . (B) Heatmap of the 15 differentially expressed genes. Red indicates higher expression, while blue indicates lower expression. (C)-(E) Bar graph of pathway enrichment analysis for the 15 differentially expressed genes. The analysis was performed using the BioPlanet 2019, (C) BioCarta 2016, and (E) Reactome pathways 2024. The y-axis lists the top 15 significantly enriched terms (p < 0.05), and the x-axis shows the –log 10 (p-value). n represents the number of experimental replicates.

Article Snippet: p75 NTR−/− (Stock No: 002213), p75 NTR flox (p75 NTRf/f ) (Stock No: 031162), and paired related homeobox-Cre (Prx1-Cre) (Stock No: 005584) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and housed in the animal facility at the University of California, San Francisco (UCSF) according to institutional guidelines.

Techniques: Activation Assay, Gene Expression, Derivative Assay, Expressing

Journal: Bone Reports

Article Title: p75 NTR regulates postnatal skeletal development via NGF-responsive JNK signaling

doi: 10.1016/j.bonr.2025.101854

Figure Lengend Snippet: KDM4B regulates p75 NTR -mediated osteogenesis via JNK signaling pathway. (A)Western blot of JNK signaling pathway (p-JNK and p-c-Jun) in p75 NTR+/+ and p75 NTR−/− BMSCs. (B) ARS images and quantification after 21 days treatment in OIM with NGF (10 ng/ml) and/or SP600125 (5 μM). n = 4 for each group. (C) qRT-PCR analysis of epigenetic genes in p75 NTR+/+ and p75 NTR−/− BMSCs with or without NGF treatment at 2 h. n = 3 for each group. (D) ARS images and quantification after 21 days treatment in OIM with pLV-empty or pLV- Kdm4b , and NGF (10 ng/ml). n = 4 for each group. (E) Upper panel: ARS images and quantification after 21 days treatment in OIM with pLV-empty or pLV- Kdm4b in p75 NTR+/+ and p75 NTR−/− BMSCs. Lower panel: gene expression analysis of Kdm4b and Dlx5 after treating with pLV-empty or pLV- Kdm4b . Dlx5 expression increased along with the upregulation of Kdm4b . n = 4 for each group. * p < 0.05, ** p < 0.01, *** p < 0.001. Two-way ANOVA with multiple comparison was used for c, d and e, followed by Tukey's post-hoc test. n represents the number of experimental replicates.

Article Snippet: p75 NTR−/− (Stock No: 002213), p75 NTR flox (p75 NTRf/f ) (Stock No: 031162), and paired related homeobox-Cre (Prx1-Cre) (Stock No: 005584) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and housed in the animal facility at the University of California, San Francisco (UCSF) according to institutional guidelines.

Techniques: Western Blot, Quantitative RT-PCR, Gene Expression, Expressing, Comparison

Journal: Oncotarget

Article Title: CD271 is a functional and targetable marker of tumor-initiating cells in head and neck squamous cell carcinoma

doi:

Figure Lengend Snippet: Expression of CD271 in human primary SCCHN samples measured by immunohistochemistry

Article Snippet: Antibody to mouse CD271 (mu p75) was obtained from Advanced Targeting Systems.

Techniques: Expressing

Journal: Oncotarget

Article Title: CD271 is a functional and targetable marker of tumor-initiating cells in head and neck squamous cell carcinoma

doi:

Figure Lengend Snippet: (A) Human primary SCCHN samples were stained with a monoclonal antibody against CD271 and assessed by immunohistochemistry and flow cytometry. The FACS plot is gated on the DAPI − lineage − population and co-stained for CD44 and CD271. (B) Two murine cell lines derived from oral tumors arising from DMBA-treated oral cavity mucosa were stained with a monoclonal antibody specific for murine CD271 and assessed by FACS. (C) Human cell lines derived from head and neck SCC were assessed for CD271 expression by FACS after staining with a monoclonal antibody to CD271.

Article Snippet: Antibody to mouse CD271 (mu p75) was obtained from Advanced Targeting Systems.

Techniques: Staining, Immunohistochemistry, Flow Cytometry, Derivative Assay, Expressing

Journal: Oncotarget

Article Title: CD271 is a functional and targetable marker of tumor-initiating cells in head and neck squamous cell carcinoma

doi:

Figure Lengend Snippet: Xenograft tumor formation with sorted population of human and mouse SCCHN cells

Article Snippet: Antibody to mouse CD271 (mu p75) was obtained from Advanced Targeting Systems.

Techniques: Injection

Journal: Oncotarget

Article Title: CD271 is a functional and targetable marker of tumor-initiating cells in head and neck squamous cell carcinoma

doi:

Figure Lengend Snippet: (A) Four cell lines derived from human SCCHN were assessed for in vitro growth kinetics by MTT assay and for in vivo tumor formation and growth by implantation into the flanks of Rag −/− γc −/− mice. (B) Knockdown of CD271 in human SCCHN cell lines by shRNA lentiviral constructs was assessed by FACS. (C) Effects of CD271 knockdown by shRNA expression on cell proliferation was assessed by MTT assay. In the cell lines with high expression of CD271 (UPCI:SCC-103 and PCI-13), there was a decrease in cell viability at the end of the MTT assay. Two different shRNAs were assessed: “sh3” = CD271 shRNA3. “sh5” = CD271 shRNA5. (D) The effects of the CD271 knockdown was reversed by co-expression of the CD271 coding sequence that lacked the 3′-UTR, which was targeted by the shRNA. In the FACS plots, the rescued expression of CD271 is demonstrated in the plot on the right. The bar graph represents the fold change in cell viability at the end of an MTT assay (relative to day 0). “c” = empty vector control or scramble RNA control. “p75”= CD271 coding sequence lacking the 3′-UTR. “sh3” = CD271 shRNA3. “sh5” = CD271 shRNA5 *p<0.01, **p<0.005. (E) PCI-13 cells were transduced with lentivirus expressing CD271 shRNA3, CD271 shRNA5, or a scrambled control RNA. Percentages of cells in the various phases of the cell cycle were assessed by FACS after staining with BrdU and PI; these percentages are quantified in the bar graph. *p<0.01.

Article Snippet: Antibody to mouse CD271 (mu p75) was obtained from Advanced Targeting Systems.

Techniques: Derivative Assay, In Vitro, MTT Assay, In Vivo, Knockdown, shRNA, Construct, Expressing, Sequencing, Plasmid Preparation, Control, Transduction, Staining

Journal: Oncotarget

Article Title: CD271 is a functional and targetable marker of tumor-initiating cells in head and neck squamous cell carcinoma

doi:

Figure Lengend Snippet: (A) Schematic of in vivo competition assay. PCI-13 cells were transduced with either a lentivirus co-expressing shRNA targeting CD271 and mCherry or a lentivirus expressing Zsgreen. Cells were mixed 1:1 and injected into the flanks of Rag −/− γc −/− mice. Cell composition in tumors arising in these mice was assessed by FACS analysis of cells from dissociated tumors. (B) FACS analysis of input cells at time of implantation and cells from dissociated tumors at the termination of the experiment. The bar graphs depict the percentages of mCherry and Zsgreen expressing tumor cells from each cohort of recipient mice.

Article Snippet: Antibody to mouse CD271 (mu p75) was obtained from Advanced Targeting Systems.

Techniques: In Vivo, Competitive Binding Assay, Transduction, Expressing, shRNA, Injection

Journal: Oncotarget

Article Title: CD271 is a functional and targetable marker of tumor-initiating cells in head and neck squamous cell carcinoma

doi:

Figure Lengend Snippet: (A) PCI-13 cells were incubated with either azide-free monoclonal antibody specific for CD271 or isotype control IgG for 30 min, washed, and then assessed for cell proliferation in vitro and tumor growth in vivo . The graph on the left shows MTT cell viability results over 8 days, expressed as fold-change from the day 0 baseline. The graph on the right shows tumor growth from cells injected subcutaneously into the flanks of Rag −/− γc −/− mice. Each cohort consists of 4 mice. Experiments were performed at least two times. (B) PCI-13 cells (grown in serum-free medium for 24 hrs) were incubated in vitro with monoclonal antibody to CD271 or isotype control IgG for 30 min and then with or without recombinant human NGF for 1 hr. Cell lysates were subjected to gel electrophoresis, and Western immunoblot analysis was performed with antibody specific for phosphorylated-Erk (p-Erk1/2), total Erk, and actin. All experiments performed two or more times.

Article Snippet: Antibody to mouse CD271 (mu p75) was obtained from Advanced Targeting Systems.

Techniques: Incubation, Control, In Vitro, In Vivo, Injection, Recombinant, Nucleic Acid Electrophoresis, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Characterization of TNF receptor subtype expression and signaling on pulmonary endothelial cells in mice

doi: 10.1152/ajplung.00326.2010

Figure Lengend Snippet: Flow cytometric analysis of TNF receptor (TNFR) expression on pulmonary endothelial cells (PECs). A: in characterization studies, PECs were identified as PECAM-1+ events (R1), and their endothelial phenotype was confirmed by positive staining for endothelial cell markers, i.e., endoglin (CD105), VE-cadherin (CD144), ICAM-2 (CD102), mucosialin (CD34), and Griffonia simplicifolia lectin. Staining with the respective antibody (solid line) and isotype control (unstained in the case of lectin; shaded fill) is shown on histograms. B: in control condition, PECs showed greater expression of p55 (open bar) than p75 (solid bar). Values are means + SD; N = 4. ***P < 0.001 by paired t-test. C: liver endothelial cells expressed similar amount of the p55 (open bar) and p75 (solid bar) receptors. Values are means + SD; N = 3. D: validation of flow cytometry technique using wild-type (WT) and p55 and p75 knockout animals (TNFRp55KO and TNFRp75KO, respectively) indicated small nonspecific binding of the p75 antibody. Staining with the respective antibody (solid line) and isotype control (shaded fill) is shown on histograms. MFI, mean fluorescence intensity; SSC, side scatter.

Article Snippet: Experiments were performed using male wild-type C57/Bl6 mice, aged 9–12 wk, or age- and sex-matched mice lacking p55 [p55 knockout (p55KO)], p75 (p75KO), or both [double knockout (DKO)] TNF receptors (generous gifts from Amgen, Thousand Oaks, CA) ( 50 ).

Techniques: Expressing, Staining, Control, Biomarker Discovery, Flow Cytometry, Knock-Out, Binding Assay, Fluorescence

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Characterization of TNF receptor subtype expression and signaling on pulmonary endothelial cells in mice

doi: 10.1152/ajplung.00326.2010

Figure Lengend Snippet: Effects of chronic absence of TNFR subtype signaling on TNF-induced expression of adhesion molecules on PECs in vivo. Surface expression of VCAM-1 (A), E-selectin (B), and ICAM-1 (C) was assessed by flow cytometry on PECs in different mouse strains either 2 (E-selectin) or 4 h (VCAM-1, ICAM-1) after intravenous (iv) TNF injection (500 ng). p75KO, TNFRp75 knockout; p55KO, TNFRp55 knockout; DKO, TNFRp55/p75 double knockout. Values are means + SD of MFI; N = 3–6/group. **P < 0.01, ***P < 0.001 vs. TNF-treated WT by one-way ANOVA with Bonferroni posttests.

Article Snippet: Experiments were performed using male wild-type C57/Bl6 mice, aged 9–12 wk, or age- and sex-matched mice lacking p55 [p55 knockout (p55KO)], p75 (p75KO), or both [double knockout (DKO)] TNF receptors (generous gifts from Amgen, Thousand Oaks, CA) ( 50 ).

Techniques: Expressing, In Vivo, Flow Cytometry, Injection, Knock-Out, Double Knockout

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Characterization of TNF receptor subtype expression and signaling on pulmonary endothelial cells in mice

doi: 10.1152/ajplung.00326.2010

Figure Lengend Snippet: Effects of acute TNFR inhibition on TNF-induced expression of adhesion molecules in primary PEC culture. Fold changes (compared with untreated cells in the same experiment) are shown of adhesion molecule expressions on WT primary mouse lung endothelial cells following 4-h TNF treatment (10 ng/ml) in the presence or absence of p75 (20 μg/ml; A) or p55 (90 μg/ml; B) receptor neutralizing monoclonal antibody (MAb). p55 and p75 blockade experiments were conducted separately. Values are means + SD; N = 3–4. *P < 0.05, **P < 0.01, ***P = 0.001 vs. TNF by paired t-test.

Article Snippet: Experiments were performed using male wild-type C57/Bl6 mice, aged 9–12 wk, or age- and sex-matched mice lacking p55 [p55 knockout (p55KO)], p75 (p75KO), or both [double knockout (DKO)] TNF receptors (generous gifts from Amgen, Thousand Oaks, CA) ( 50 ).

Techniques: Inhibition, Expressing

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Characterization of TNF receptor subtype expression and signaling on pulmonary endothelial cells in mice

doi: 10.1152/ajplung.00326.2010

Figure Lengend Snippet: Changes in TNFR expression on PECs during endotoxemia in vivo. Mice were injected with iv LPS (20 μg), and after 2, 4, 8, and 24 h, the lungs were processed for flow cytometric analysis. Untreated animals served as controls. Surface expression of TNFR p55 and p75 was quantified on PECAM-1+ events, as described before. Values are means ± SD of MFI; N = 4–5/each time point. ***P < 0.001 vs. control for p55 expression by one-way ANOVA with Bonferroni posttests.

Article Snippet: Experiments were performed using male wild-type C57/Bl6 mice, aged 9–12 wk, or age- and sex-matched mice lacking p55 [p55 knockout (p55KO)], p75 (p75KO), or both [double knockout (DKO)] TNF receptors (generous gifts from Amgen, Thousand Oaks, CA) ( 50 ).

Techniques: Expressing, In Vivo, Injection, Control

Journal: The Journal of Neuroscience

Article Title: p75 Neurotrophin Receptor Expression Defines a Population of BDNF-Responsive Neurogenic Precursor Cells

doi: 10.1523/JNEUROSCI.0654-07.2007

Figure Lengend Snippet: Neurosphere size is unaffected by p75 NTR expression or BDNF treatment

Article Snippet: Scale bars, 50 μm. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2. caption a7 Purification of p75 NTR -positive cells from rat SVZ. a , b , Freshly isolated and dissociated P2 ( a ) and adult ( b ) rat SVZ cells were immunostained with the mouse anti-rat extracellular p75 NTR antibody (Millipore) and sorted by flow cytometry into three populations, as shown in the representative profiles. p75 NTR -high, -mid, and -negative cell regions are indicated by the red, green, and blue boxes, respectively.

Techniques: Expressing, Negative Control