Journal: Oncogene

Article Title: Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

doi: 10.1038/onc.2009.446

Figure Lengend Snippet: Figure 1 c-Cbl ubiquitin ligase regulates proteasomal degradation of Notch3-IC. (a) Co-transfection of HEK293 cells with flag Notch3-IC and HA-c-Cbl expression plasmids. Whole lysates were immunoprecipitated with anti-flag antibody and revealed in western blot with anti-Ub and anti-HA antibodies. Anti-flag antibody was used as a control of the immunoprecipitation assay. Total lysates were analyzed for the expression of c-Cbl plasmid using anti-HA antibody. (b, c) Co-transfection of HEK293 cells with flag Notch3-IC and HA c-Cbl expression plasmids: cells were treated with (b) proteasome inhibitor MG132 in a time course study for 0–5 h or with (c) cycloheximide (CHX) and/or MG132 for 5 h before lysis. In both experiments, whole lysates obtained were revealed in western blot with anti-flag antibody, followed by anti-b-actin antibody used as a control of sample loading (left panels). The right panels show the relative quantification as determined by optical densitometry (OD).

Article Snippet: Thymocytes were stained with Alexa Fluor 594-conjugate CTB (Molecular Probes, Leiden, The Netherlands) at a final concentration of 40 mg/ml, for 40min at þ 4 1C, followed by anti-Notch3 antibody (M-134; Santa Cruz Biotechnology), as previously described (Felli et al., 2005).

Techniques: Ubiquitin Proteomics, Cotransfection, Expressing, Immunoprecipitation, Western Blot, Control, Plasmid Preparation, Lysis

Journal: Oncogene

Article Title: Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

doi: 10.1038/onc.2009.446

Figure Lengend Snippet: Figure 3 Protein kinase C y (PKCy) regulates c-Cbl phosphorylation status in ex vivo and in vitro experiments, resulting in the regulation of Notch3-IC protein levels. (a) Whole-cell extracts of thymocytes from wt, tg N3-IC and N3-IC/pTa/ mice were immunoprecipitated with anti-c-Cbl antibody and revealed in western blot with anti-P-tyr antibody, stripped and reprobed with anti-P- Ser PKC substrate antibody. Anti-c-Cbl immunoblotting was used as a control of the immunoprecipitation assay. Western blot analysis of c-Cbl in total lysates shows that the modification of c-Cbl phosphorylation is not accounted for by changes of its protein levels. Data are representative of three similar experiments. (b) Treatment of N3-IC/pTa/ double-mutant thymocytes with phorbol 12-myristate 13-acetate (PMA) for 0–15–30 min before lysis: total lysates were revealed in western blot with anti-P-tyr and anti-Cbl antibodies. Molecular weight markers are shown on the left (left panel). The same samples were immunoprecipitated with anti-c-Cbl antibody and revealed in western blot with anti-P-tyr antibody, stripped and reprobed with anti-P-Ser PKC substrate and anti-PKCy antibodies. Anti-c-Cbl immunoblotting was used as a control of the immunoprecipitation assay (right panel). (c) Treatment of N3-IC/ pTa/ double-mutant thymocytes with PMA for 3 h before lysis: western blot analysis of Notch3 protein levels. Anti-b-actin immunoblotting was used as a control of sample loading. (d) Co-transfection of HEK293 cells with HA-c-Cbl plasmid in presence or absence of DN-PKCy and CA-PKCy expression vectors. Whole lysates were immunoprecipitated with anti-c-Cbl antibody and revealed in western blot with anti-P-tyr antibody, stripped and reprobed with anti-P-Ser and anti-PKCy antibodies. The immunoblotting with anti-Cbl antibody was used as a control of the immunoprecipitation assay. (e) Co-transfection of HEK293 cells with flag Notch3-IC and HA c-Cbl plasmids in presence or absence of DN-PKCy and CA-PKCy expression vectors. Whole lysates were immunoprecipitated with anti-flag antibody and revealed in western blot with anti-Ub antibody, followed by anti-flag antibody used as a control of the immunoprecipitation assay. The same whole lysates were immunoprecipitated with anti-c-Cbl antibody and revealed in western blot with anti-flag antibody to detect c-Cbl/Notch3 interaction. (f) HEK293 cells, co-transfected as previously described, were treated with proteasome inhibitor MG132 for 5 h before lysis. Whole lysates were revealed in western blot with anti-flag antibody, followed by anti-b-actin antibody, used as a control of sample loading.

Article Snippet: Thymocytes were stained with Alexa Fluor 594-conjugate CTB (Molecular Probes, Leiden, The Netherlands) at a final concentration of 40 mg/ml, for 40min at þ 4 1C, followed by anti-Notch3 antibody (M-134; Santa Cruz Biotechnology), as previously described (Felli et al., 2005).

Techniques: Phospho-proteomics, Ex Vivo, In Vitro, Immunoprecipitation, Western Blot, Control, Mutagenesis, Lysis, Molecular Weight, Cotransfection, Plasmid Preparation, Expressing, Transfection

Journal: Oncogene

Article Title: Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

doi: 10.1038/onc.2009.446

Figure Lengend Snippet: Figure 4 pTa directly interacts with Notch3 and regulates its recruitment to lipid rafts. (a) Whole-cell extracts, cytosol and membrane fractions and rafts and nonrafts fractions of N3-232T cells were revealed in western blot with anti-N3EC and anti-N3IC antibodies. Results are representative of three similar experiments. (b) Left panel: confocal microscopy of thymocytes from wt and tg N3-IC mice stained with Alexa Fluor 594-conjugate CTB and rabbit polyclonal anti-Notch3 antibody, as described in Materials and methods section. Right panel: rafts and nonrafts fractions derived from the same cells were revealed in western blot with anti-N3EC and anti-pTa antibodies. Anti-ZAP-70 was used to control the activation status. For T-cell activation, cells were incubated with anti-CD3 antibody as described in Materials and methods section. (c) Rafts and nonrafts fractions derived from SCB29 and SCIET27 pre-T-cell lines were revealed in western blot with anti-N3EC antibody. (d) Whole-cell extracts (left panel), rafts and nonrafts fractions (right panel) derived from tg N3-IC and N3-IC/pTa/ mice were revealed in western blot with anti-N3EC. (e) Co-transfection of HEK293 cells with HA- Notch3-IC and pTa expression plasmids. Whole lysates were immunoprecipitated with anti-pTa antibody and revealed in western blot with anti-HA antibody, followed by anti-pTa antibody, used as a control of the immunoprecipitation assay. * indicates a nonspecific band (left panel). Whole lysates from 2017 pre-T-cell line were immunoprecipitated with anti-pTa antibody and revealed in western blot with anti-N3IC antibody, followed by anti-pTa antibody, used as a control of the immunoprecipitation assay (right panel). Data are representative of three similar experiments. Anti-p56Lck and anti-tubulin immunoblotting were used as a control of fractionation; anti-b-actin immunoblotting was used to monitor sample loading. WCE, whole-cell extracts; C, cytosol; M, membrane; R, rafts; NR, nonrafts; FL, full-length receptor; EC, extracellular fragment; TM, transmembrane fragment; IC, intracellular fragment; un, untransfected cells; tr, transfected cells.

Article Snippet: Thymocytes were stained with Alexa Fluor 594-conjugate CTB (Molecular Probes, Leiden, The Netherlands) at a final concentration of 40 mg/ml, for 40min at þ 4 1C, followed by anti-Notch3 antibody (M-134; Santa Cruz Biotechnology), as previously described (Felli et al., 2005).

Techniques: Membrane, Western Blot, Confocal Microscopy, Staining, Derivative Assay, Control, Activation Assay, Incubation, Cotransfection, Expressing, Immunoprecipitation, Fractionation, Transfection

Journal: Oncogene

Article Title: Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

doi: 10.1038/onc.2009.446

Figure Lengend Snippet: Figure 5 Notch3/pTa relationship favors c-Cbl recruitment to lipid rafts. (a) Rafts and nonrafts fractions from wt thymocytes, resting or activated with anti-CD3 antibody, and tg N3-IC thymocytes, not activated, were revealed in western blot with anti c-Cbl antibody. Anti-ZAP-70 and anti-b-actin immunoblotting were used to control the activation process and to monitor sample loading, respectively. (b) Treatment of N3-232T cells with furine-like inhibitor A23187 before rafts isolation. Rafts and nonrafts fractions were revealed in western blot with anti-c-Cbl antibody. Anti-N3EC and anti-N3IC immunoblotting were used as a control of the treatment; anti-p56Lck and anti-Lat immunoblotting were used as a control of rafts aggregation process. * indicates the Notch3-IC protein. (c) Rafts and nonrafts fractions from tg N3-IC and N3-IC/pTa/ thymocytes were revealed in western blot with anti-c-Cbl antibody. Anti-p56Lck and anti-tubulin immunoblotting were used as a control of fractionation. The samples are normalized using b-actin protein expression (left panel). The right panel shows the quantification of c-Cbl protein associated to rafts in tg N3-IC and N3-IC/pTa/ thymocytes. Data represent the average of three independent experiments. (d) c-Cbl/Notch3-IC cytosolic interaction after rafts disrupting. Treatment of N3-232T cells with methyl-b-cyclodextrin (MbCD) before subcellular fractionation: cytosolic fractions derived from N3- 232T MbCD-treated and -untreated cells were used for immunoprecipitation assay with anti-c-Cbl antibody and revealed in western blot with anti-N3IC antibody, followed by anti-c-Cbl antibody, used as a control of the immunoprecipitation assay. R, rafts; I, intermediate fractions; NR, nonrafts; FL, full-length receptor; EC, extracellular fragment; TM, transmembrane fragment.

Article Snippet: Thymocytes were stained with Alexa Fluor 594-conjugate CTB (Molecular Probes, Leiden, The Netherlands) at a final concentration of 40 mg/ml, for 40min at þ 4 1C, followed by anti-Notch3 antibody (M-134; Santa Cruz Biotechnology), as previously described (Felli et al., 2005).

Techniques: Western Blot, Control, Activation Assay, Isolation, Fractionation, Expressing, Derivative Assay, Immunoprecipitation

Journal: Oncogene

Article Title: Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

doi: 10.1038/onc.2009.446

Figure Lengend Snippet: Figure 6 Hypothetical model linking c-Cbl different subcellular localization with Notch3 degradation. (a) In presence of a functional pre-TCR/Notch3 relationship, c-Cbl is able to translocate to the rafts compartment, where it is preferentially phosphorylated in serine- rich motifs through protein kinase C y (PKCy). In these conditions c-Cbl seems to lack its E3 ubiquitin ligase upon Notch3, thus sustaining the oncogenic role of Notch3 and pTa relationship with respect to T-cell leukemogenesis. (b) In the absence of pre-TCR, c-Cbl localizes preferentially in cytosolic fraction. In addition, deletion of pTa results in reduction of PKCy activity that in turn favors tyrosine phosphorylation of c-Cbl and its increased E3 ubiquitin ligase activity upon Notch3-IC, targeting it to proteasomal-degradative pathway and contributing to the rescue of T-cell leukemia. P-tyr, tyrosine phosphorylation; P-Ser, serine phosphorylation; Ub, ubiquitination.

Article Snippet: Thymocytes were stained with Alexa Fluor 594-conjugate CTB (Molecular Probes, Leiden, The Netherlands) at a final concentration of 40 mg/ml, for 40min at þ 4 1C, followed by anti-Notch3 antibody (M-134; Santa Cruz Biotechnology), as previously described (Felli et al., 2005).

Techniques: Functional Assay, Ubiquitin Proteomics, Activity Assay, Phospho-proteomics

Journal: Cell death & disease

Article Title: Genetic alterations of Keap1 confers chemotherapeutic resistance through functional activation of Nrf2 and Notch pathway in head and neck squamous cell carcinoma.

doi: 10.1038/s41419-022-05126-8

Figure Lengend Snippet: Fig. 6 Nrf2 regulates Notch signaling in HNSCC cells. A Expression of Notch1 and Notch target genes mRNA in control and Keap1-expressing SSC9 clone cells. B Expression of Notch1 and Hes1 proteins in Keap1-mutant and Keap1-expressing SSC9 cells. C Notch1 and Hes1 mRNA and, D protein expression after Nrf2 knockdown in SSC9 cells. E Immunohistochemistry staining and expression of Nrf2, Ki67, Notch1, and Hes1 in HNSCC clinical samples from wild-type, Nrf2, and Keap1 mutant patients tumor tissues. F Notch1 expression in non-targeting control and Notch1 siRNA-treated SSC9 cells G Cell proliferation of SSC9 cells after knockdown of Notch1 by siRNA. H Relative mRNA expression of Hes1 and Hey1 after Notch1 knockdown in SSC9 cells. I Hes1 mRNA expression and, J Cell proliferation after knockdown of Hes1 siRNA in SSC9 cells. K Effects of Notch inhibitor DAPT and, L Assessment of cell growth after treating the cells with Notch inhibitor DAPT for 5-days. The mRNA expression levels were calculated and normalized relative to GAPDH. All experiments were run in triplicate and compared with the control group. Data presents as mean ± SEM (**P < 0.01, ***P < 0.001).

Article Snippet: Primary antibodies are Nrf2 (Abcam, cat# ab137550, MA, USA) and Keap1 (Abcam, cat# 119403 MA, USA), Notch1 (cat# 14-5785-81) and Hes1 (cat# PA5-28802; Invitrogen, USA), and GAPDH (Santa Cruz, cat# sc32233, CA, USA).

Techniques: Expressing, Control, Mutagenesis, Knockdown, Immunohistochemistry, Staining

Journal: Cell death & disease

Article Title: Genetic alterations of Keap1 confers chemotherapeutic resistance through functional activation of Nrf2 and Notch pathway in head and neck squamous cell carcinoma.

doi: 10.1038/s41419-022-05126-8

Figure Lengend Snippet: Fig. 7 Combination therapy with cetuximab, paclitaxel, and cisplatin led to a partial response in a patient with Keap1 mutant advanced- stage metastatic HNSCC. A Clinical characteristic of head and neck cancer patient cohort treated with chemotherapy and analyzed for Keap1 and Nrf2 mutation by Sanger sequencing. B, C Association Keap1 mutations and local treatment failure in patients with HNSCC treated with chemotherapy. B Patient cohort and, C Stage III–IV patients who were treated with chemotherapy. D, E Tumor progression in an index patient with lung metastasis was associated with the identification of Keap1 and Shh mutations and tumors from index patients with Keap1 mutant strongly expressed Notch1 and Hes1. In both cases, patients with Keap1 mutations achieved a partial response to 31% and 37% reduction, respectively, in the metastatic lung region upon treatment with two lines of chemoradiation/cetuximab (patient case #1) and three cycles of TPE (docetaxel, cisplatin, and fluorouracil (TPF) followed by chemoradiation with cisplatin treatment (patient case #2). F Clinical courses/ outcomes of Keap1 mutant HNSCC patient treated with chemoradiation therapy. SD stable disease, PD progressive disease.

Article Snippet: Primary antibodies are Nrf2 (Abcam, cat# ab137550, MA, USA) and Keap1 (Abcam, cat# 119403 MA, USA), Notch1 (cat# 14-5785-81) and Hes1 (cat# PA5-28802; Invitrogen, USA), and GAPDH (Santa Cruz, cat# sc32233, CA, USA).

Techniques: Mutagenesis, Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Activates Notch4 via ERK/JNK/P38 MAPK Signaling Pathways to Promote Lung Adenocarcinoma Progression and Metastasis

doi: 10.3389/fcell.2021.780121

Figure Lengend Snippet: Notch4 is upregulated in LUAD tissues and cells. (A) The protein level of Notch4 in five paired LUAD tissues and adjacent non-malignant tissues. (B) The protein levels of Notch4 in A549 and H1299 cells and normal HBE cell. ( n = 8). (C) The protein levels of Notch4 in H1299 cells exposed to hypoxia for different times. ( n = 3). (D) The protein levels of Notch4 in A549 cells exposed to hypoxia for different times. ( n = 5). Data were presented as means ± SD. * p < 0.05, ** p < 0.01. LUAD, lung adenocarcinoma; HBE, human bronchial epithelial.

Article Snippet: Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Activates Notch4 via ERK/JNK/P38 MAPK Signaling Pathways to Promote Lung Adenocarcinoma Progression and Metastasis

doi: 10.3389/fcell.2021.780121

Figure Lengend Snippet: Notch4 gene silencing alleviated xenograft tumor growth and metastasis. (A–F) Xenograft tumor growth experiments were performed in nude mice with A549-control and A549-shNotch4 stable cells. (A) Representative images of subcutaneous tumor dissected from the nude mice were presented. (B) Representative H&E staining images in sections of tumor were presented. Magnification, ×200; Bar, 50 μm. (C) Subcutaneous tumor growth curves of the nude mice were presented. ( n = 10). (D) Subcutaneous tumor weights were presented. ( n = 10). (E) The mRNA level of Notch4 in xenograft tumor. ( n = 8–10). (F) The protein levels of Notch4, p38, p-P38, JNK, p-JNK, ERK, p-ERK in xenograft tumor. ( n = 3). (G, I) Lung metastasis experiments were performed in nude mice with A549-control and A549-shNotch4 stable cells. (G) Representative images of lung metastases were presented. (H) The numbers of visible metastatic nodules in the lungs of mice were counted. ( n = 3–4). (I) Representative H&E staining images in sections of lung tissues were presented. Magnification, ×200; Bar, 50 μm. Data were presented as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. H&E, hematoxylin-eosin staining. JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase.

Article Snippet: Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Control, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Activates Notch4 via ERK/JNK/P38 MAPK Signaling Pathways to Promote Lung Adenocarcinoma Progression and Metastasis

doi: 10.3389/fcell.2021.780121

Figure Lengend Snippet: Notch4 is involved in the regulation of cell proliferation in A549 and H1299 cells exposed to hypoxia. (A, B) Cell viability was assessed using CCK-8 assay in A549 and H1299 cells ( n = 4–5). (C, D) A549 cell proliferation was assessed using Edu assay. ( n = 4). Magnification, ×400; Bar, 20 μm. (E, F) H1299 cell proliferation was assessed using Edu assay. ( n = 5). Magnification, ×400; Bar, 20 μm. (G) Colony formation assay was performed in A549 and H1299 cells. ( n = 3). Data were presented as means ± SD. * p < 0.05, comparison with normoxic cells treated with si-NC; # p < 0.05, comparison with hypoxic cells treated with si-NC. CCK-8, Cell Counting Kit-8; si-NC, negative control short interfering RNAs (siRNA); si-Notch4, the siRNA against Notch4.

Article Snippet: Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: CCK-8 Assay, EdU Assay, Colony Assay, Comparison, Cell Counting, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Activates Notch4 via ERK/JNK/P38 MAPK Signaling Pathways to Promote Lung Adenocarcinoma Progression and Metastasis

doi: 10.3389/fcell.2021.780121

Figure Lengend Snippet: Notch4 is involved in the regulation of cell migration and apoptosis in A549 and H1299 cells exposed to hypoxia. (A) Wound healing assay was performed in A549 and H1299 cells. ( n = 4). Magnification, ×40; Bar, 200 μm. (B) Cell apoptosis was assessed by Annexin-V/PI staining. ( n = 3). Analyses of apoptosis including early apoptosis (Annexin-V positive and PI negative) and late apoptosis (Annexin-V positive and PI positive) were shown. Data were presented as means ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001, comparison with normoxic cells treated with si-NC; # p < 0.05, comparison with hypoxic cells treated with si-NC. si-NC, negative control short interfering RNAs (siRNA); si-Notch4, the siRNA against Notch4; PI, propidium iodide.

Article Snippet: Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Migration, Wound Healing Assay, Staining, Comparison, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Activates Notch4 via ERK/JNK/P38 MAPK Signaling Pathways to Promote Lung Adenocarcinoma Progression and Metastasis

doi: 10.3389/fcell.2021.780121

Figure Lengend Snippet: ERK, JNK, and P38 MAPK signaling mediate the regulation of Notch4 overexpression on A549 and H1299 cells proliferation and apoptosis. (A) The MAPK signaling pathway were detected in A549 and H1299 cells transfected with siRNA against Notch4 or negative control siRNA. (B) Cell viability were examined in A549 and H1299 cells transfected with Notch4 plasmid or negative control plasmid co-treatment with ERK (U0126), JNK (SP600125), or P38 (SB203580) MAPK pathway inhibitors by using CCK-8 assay. ( n = 3–4). (C) Colony formation assay was performed in A549 and H1299 cells transfected with Notch4 plasmid or negative control plasmid co-treatment with U0126, SP600125, or SB203580 for 24 h ( n = 4). (D) The Annexin-V/PI assay was performed in A549 and H1299 cells transfected with Notch4 plasmid or negative control plasmid co-treatment with U0126, SP600125, or SB203580 for 24 h ( n = 3–6). Analyses of apoptosis including early apoptosis (Annexin-V positive and PI negative) and late apoptosis (Annexin-V positive and PI positive) were shown. Data were presented as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. CCK-8, Cell Counting Kit-8; PI, propidium iodide; si-NC, negative control short interfering RNAs (siRNA); si-Notch4, the siRNA against Notch4. Vector, negative control plasmid; Notch4, Notch4 plasmid. ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase.

Article Snippet: Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Over Expression, Transfection, Negative Control, Plasmid Preparation, CCK-8 Assay, Colony Assay, Cell Counting

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Activates Notch4 via ERK/JNK/P38 MAPK Signaling Pathways to Promote Lung Adenocarcinoma Progression and Metastasis

doi: 10.3389/fcell.2021.780121

Figure Lengend Snippet: ERK, JNK, and P38 MAPK signaling mediate the regulation of Notch4 overexpression on A549 and H1299 cells migration. (A–D) Wound healing assay was performed in A549 and H1299 cells transfected with Notch4 plasmid or negative control plasmid co-treatment with U0126, SP600125, or SB203580. ( n = 3). Magnification, ×40; Bar, 200 μm. (E–H) Transwell migration assay was performed in A549 and H1299 cells transfected with Notch4 plasmid or negative control plasmid co-treatment with U0126, SP600125, or SB203580 for 24 h ( n = 3). Magnification, ×100; Bar, 50 μm. Data were presented as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Vector, negative control plasmid; Notch4, Notch4 plasmid. ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase.

Article Snippet: Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Over Expression, Migration, Wound Healing Assay, Transfection, Plasmid Preparation, Negative Control, Transwell Migration Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Activates Notch4 via ERK/JNK/P38 MAPK Signaling Pathways to Promote Lung Adenocarcinoma Progression and Metastasis

doi: 10.3389/fcell.2021.780121

Figure Lengend Snippet: Notch4 interacts with ERK, JNK, and P38 in A549 cells. Endogenous co-immunoprecipitation was performed in A549 cells exposed to normoxia or hypoxia for 24 h. (A) Western blot analysis for endogenous ERK, JNK, and P38 after IP of endogenous Notch4 from A549 cells exposed to normoxia or hypoxia. (B) Western blot analysis for endogenous Notch4 after IP of endogenous ERK from A549 cells exposed to normoxia or hypoxia. (C) Western blot analysis for endogenous Notch4 after IP of endogenous JNK from A549 cells exposed to normoxia or hypoxia. (D) Western blot analysis for endogenous Notch4 after IP of endogenous P38 from A549 cells exposed to normoxia or hypoxia. (E) Whole-cell lysates were used for IB with the indicated antibodies to show expression. IP, immunoprecipitation; IB, immunoblot. ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.

Article Snippet: Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Immunoprecipitation, Western Blot, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxia Activates Notch4 via ERK/JNK/P38 MAPK Signaling Pathways to Promote Lung Adenocarcinoma Progression and Metastasis

doi: 10.3389/fcell.2021.780121

Figure Lengend Snippet: Schematic illustration of the role of Notch4 in the proliferation, apoptosis and migration of LUAD cells. Hypoxia induces the expression of Notch4. Increased level of Notch4 promotes cell proliferation, migration, and apoptosis resistance in A549 and H1299 cells. Hypoxia activates Notch4 via ERK/JNK/P38 MAPK signaling pathways to promote lung adenocarcinoma progression and metastasis. LUAD, lung adenocarcinoma; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase.

Article Snippet: Primary antibody against Notch4 was obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Migration, Expressing, Protein-Protein interactions

Journal: The Journal of Experimental Medicine

Article Title: Inherited IL-18BP deficiency in human fulminant viral hepatitis

doi: 10.1084/jem.20190669

Figure Lengend Snippet: Homozygous 40-nt deletion in IL18BP . (A) Pedigree of the family affected by FVH due to HAV. The patient is shown in black, whereas healthy individuals are shown in white. Where available, IL18BP mutation (NM_173042.2:c.508-19_528del) status is indicated in red. M, mutant. (B) Familial segregation of the mutation and its homozygous state in the patient were confirmed by Sanger sequencing. (C) Graph showing the predicted CADD scores and global AFs of the mutation found in the patient with FVH (red circle) and missense variants of IL18BP (blue circles) for which homozygotes were reported in GnomAD. The CADD-MSC score (90% confidence interval) for IL18BP is indicated by a dashed line. (D) The upper panel shows the exons (1–5) of the canonical IL18BP transcript; the bottom panel shows a diagram for IL-18BP. The signal peptide is highlighted in blue; the Ig domain is shown in red. Start and stop codons are indicated by an arrow and an asterisk, respectively. The c.508-19_528del is shown as a dashed box on the mRNA. The locations of IL18BP alleles from GnomAD are also shown on the protein diagram.

Article Snippet: Immunoblotting was performed with primary antibodies against the His-Tag (MA1-21315, 0.5 μg/ml; Thermo Fisher Scientific) and IL-18BP (AF119, 0.5 μg/ml; R&D Systems).

Techniques: Mutagenesis, Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Inherited IL-18BP deficiency in human fulminant viral hepatitis

doi: 10.1084/jem.20190669

Figure Lengend Snippet: Impact of the IL18BP :c.508-19_528del on gene expression and function. (A) RT-qPCR showing IL18BP levels normalized against endogenous GAPDH expression in EBV-B cell lines from six healthy controls (black), the WT sibling (III.3, purple), and heterozygous family members: brother (III.2, red), father (II.4, blue), and mother (II.9, green). Relative IL18BP expression was determined by normalization against the mean value for WT cells, set to 1 (indicated by a dashed line). The values shown are the means of two independent experiments performed in duplicate. (B) Agarose gel electrophoresis showing aberrant splicing of the IL18BP mRNA in 3′ RACE on EBV-B cells from the heterozygous sibling (III.2), relative to a control cell line (C1) and the WT sibling (III.3). HPRT1 was used as the housekeeping gene control. (C) The nested PCR products from B were cloned, and colonies were sequenced. Diagram (left) and percentages (right) of WT (gray) and mutant (M1 in blue, M2 in red, and M3 in green) splice variants of the IL18BP transcript are shown. The start codon is located at position 1, and the stop codon is at 585, shown by an asterisk, on the WT transcript. The polyadenylation site is at position 1,252 and indicated by A n . (D) Expression levels of each splice variant (WT in gray, M1 in blue, M2 in red, and M3 in green) were determined and normalized against endogenous GAPDH expression levels by RT-qPCR on EBV-B cells from two healthy controls (C2 and C3) and family members. Graph shows the copy numbers of the mutant splice variants relative to the mean copy number for the WT allele in EBV-B cells from C2, C3, and III.3, which was set to 1 (indicated by a dashed line). The values are the means ± SEM of two independent experiments performed in duplicate. (E and F) Representative immunoblot images showing levels of the WT and mutant IL-18BP alleles, M1–M3 (E), and four missense alleles from GnomAD (F) in concentrated supernatants from transiently transfected COS7 cells. Immunoblotting was performed with the His tag antibody (top), and the membrane was then stripped and probed with the IL18BP antibody (bottom). (G) IL-18BP bioassay: IFN-γ production was measured in NK-92 cells stimulated with recombinant human IL-12 (100 pg/ml), IL-18 (10 ng/ml), and/or concentrated supernatants (100 µg/ml of total protein) of COS7 cells transiently transfected with either empty vector or the constructs expressing indicated IL-18BP variants. Graph is presented on a logarithmic scale with base of 10. The data are the means ± SEM of two independent experiments performed in duplicate using the supernatants shown in E and F and Fig. S1, F and G.

Article Snippet: Immunoblotting was performed with primary antibodies against the His-Tag (MA1-21315, 0.5 μg/ml; Thermo Fisher Scientific) and IL-18BP (AF119, 0.5 μg/ml; R&D Systems).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Agarose Gel Electrophoresis, Control, Nested PCR, Clone Assay, Mutagenesis, Variant Assay, Western Blot, Transfection, Membrane, Bioassay, Recombinant, Plasmid Preparation, Construct

Journal: The Journal of Experimental Medicine

Article Title: Inherited IL-18BP deficiency in human fulminant viral hepatitis

doi: 10.1084/jem.20190669

Figure Lengend Snippet: Liver immunohistochemical profile of the patient. Liver tissue sections from a control individual, an unrelated patient with FVH due to HAV, and the deceased IL-18BP–deficient FVH patient reported in this study were subjected to immunohistochemical staining with the following markers: Hep Par-1, CD8, perforin, CD57, CD68, and IL-18. Representative zoom-in views of the original images at 400× magnification (Fig. S4) are shown. Hep Par-1 staining of IL-18BP–deficient patient’s liver tissue section displayed a background staining of macrophages, with lower intensity than hepatocytes. Some IL-18–positive hepatocytes and macrophages are indicated with blue and red arrows, respectively. Scale bar represents 50 µm.

Article Snippet: Immunoblotting was performed with primary antibodies against the His-Tag (MA1-21315, 0.5 μg/ml; Thermo Fisher Scientific) and IL-18BP (AF119, 0.5 μg/ml; R&D Systems).

Techniques: Immunohistochemical staining, Control, Staining

Journal: The Journal of Experimental Medicine

Article Title: Inherited IL-18BP deficiency in human fulminant viral hepatitis

doi: 10.1084/jem.20190669

Figure Lengend Snippet: IL-18/IL-18BP–mediated hepatotoxicity. (A and B) Coculture of mock- or HAV-infected hepatocytes (HepG2 and Huh7.5 cells) with NK-92 cells pretreated with IL-18, IL-18 + IL-18BP, or IL-18BP. HAV infection efficiencies in HepG2 and Huh7.5 cells were ∼40% and ~100%, respectively (Fig. S5 E; Materials and methods). The relative survival of calcein-AM–stained HepG2 or Huh7.5 cells was calculated based on the measurement of fluorescence retention within cells (A) and the amount of secreted albumin (B). Relative fluorescence and albumin levels were determined by normalization against the mean value for hepatocytes cocultured with NK92 cells without pretreatment (not treated [NT]), set to 100. A decrease in the fluorescence or in albumin levels indicates an increase in NK cell–induced hepatotoxicity. The data shown are the means ± SEM of three independent experiments performed in quadruplicate (n.s., not significant; **, P < 0.01; ***, P < 0.001; one-way ANOVA with Bonferroni correction for multiple comparisons). (C) A proposed model for IL-18BP deficiency underlying FVH. During the course of acute HAV infection in an otherwise healthy individual (left), IL-18 is secreted by macrophages in the liver. This cytokine activates lymphocytes, such as NK cells, inducing IFN-γ production and cytotoxicity to eliminate HAV-infected cells. IFN-γ also induces IL-18BP secretion by hepatocytes, macrophages, and other nonparenchymal cells (endothelial cells, fibroblasts, and hepatic stellate cells), to buffer IL-18 activity. However, in the absence of IL-18BP (right), excessive IL-18 activity leads to uncontrolled, massive immune-mediated hepatotoxicity and severe liver injury, as in the IL-18BP–deficient individual with FVH.

Article Snippet: Immunoblotting was performed with primary antibodies against the His-Tag (MA1-21315, 0.5 μg/ml; Thermo Fisher Scientific) and IL-18BP (AF119, 0.5 μg/ml; R&D Systems).

Techniques: Infection, Staining, Fluorescence, Activity Assay

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. Time- and dose-dependent inhibition of NAC on the intracellular domain of Notch3 (N3IC), but not Notch1 (N1IC). HeLa cells were treated with NAC (2-10 mM) for 0-24 h. B. Dose-dependent inhibition by NAC (0-10 mM, 6 h) on the protein expression of N3IC in HeLa cells. C. NAC treatment (5 mM, 0-12 h) reduces protein levels of N3IC and extracellular domain of Notch 3 (N3EC) but not full length Notch 3 precursor (N3FL) in HeLa cells. Densitometry quantifications of the protein bands were shown after normalization with their respective β-actin levels. Data are presented as means ± SE, n=3. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Inhibition, Expressing

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. NAC treatment (2-10 mM, 0-24 h) decreases Hes1 and HRT1 protein levels in HeLa cells. B. NAC treatment (0-10 mM for 6 h or 5 mM for 0-12 h) decreases Hes1 and HRT1 mRNA expression in HeLa cells. The mRNA expression of NAC-treated cells was normalized to that of non-treated cells whose value was set as 1. C. NAC treatment (0-10 mM, 12 h) inhibits Hes1 reporter activity in HeLa cells. The luciferase activity in NAC-treated cells was normalized to that of non-treated cells whose value was set as 1. D. Notch3 siRNA knockdown reduces Hes1 and HRT1 levels in HeLa cells. Protein levels were determined 2 days after siRNA transfection. siCtrl, scramble siRNA; siNotch3, Notch3 siRNA. Protein densitometry quantifications were shown after normalization with β-actin levels. Data are presented as means ± SE, n=3-4. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Activity Assay, Luciferase, Knockdown, Transfection

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. Pre-treatment with a γ-secretase inhibitor, DAPT (20 μM, 30 min), had no effect on NAC-induced (5 mM, 0-12 h) decrease in N3IC protein expression in HeLa cells. B. NAC treatment (5 mM, 0-12 h) did not affect Notch3 mRNA expression in HeLa cells. C. Pre-treatment with NH 4 Cl (25 mM, 1 h), but not lactacystin (10 μM, 30 min), reversed NAC-induced (5 mM, 0-12 h) decrease of N3IC protein levels in HeLa cells. D. NAC treatment did not affect levels of exogenously expressed Notch3 active intracellular domain (N3ICD). HeLa cells were transfected with vectors expressing N3ICD or N3FL for 24 h, followed by treatment with NAC (5 mM, 0-12 h). E. Subcellular analysis of Notch3 protein levels following NAC treatment (5 mM, 6 h) in HeLa cells. Protein levels of N3FL, N3EC and N3IC in cytosolic, nuclear and membrane fractions were determined. Successful fractionation was evidenced by using the marker proteins GAPDH, cyclin B1, and Na + , K + -ATPase. N3FL, N3EC and N3IC denoted Notch3 full length, extracellular domain and intracellular domain, respectively. Protein densitometry quantifications were shown after normalization with β-actin (A, C, D) or their respective cellular compartment markers (E). Data are presented as means ± SE, n=3-4. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Transfection, Membrane, Fractionation, Marker

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: N3ICD overexpression rescues NAC-induced inhibition of proliferation (A), migration (B), and invasion (C). A. Numbers of EV- and N3ICD-transfected cells were counted at 12-48 h after NAC treatment (0-10 mM, left panel). *, p < 0.05 compared with the EV-transfected cells within the same treatment and time point. B. Results of the wound healing assay (left panels) were expressed as the migration index (the distance migrated relative to the initial scraped gap) and that of EV-transfected cells without NAC treatment was set as 100% (middle panel). C. Cells per field on the insert membrane were imaged (left panels) and counted (middle panel). B and C: *, p < 0.05 compared with no NAC treatment; #, p < 0.05 compared with the EV-transfected cells within the same treatment. Percent rescue (A-C, right panels) after N3ICD expression was calculated by dividing the net change after NAC treatment in N3ICD-transfected cells by that in EV-transfected cells. Notch3 siRNA knockdown inhibits cell proliferation D. , migration E. , and invasion F. as assessed by the same approaches described above. Representative images for migration and invasion were shown. *, p < 0.05 compared with the siCtrl-transfected cells. All data are presented as mean ±SE, n=3. I, the initial seeded cell number. EV, empty vector; N3ICD, Notch3 active intracellular domain; siCtrl, scrambled siRNA; siNotch3, Notch3 siRNA.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Over Expression, Inhibition, Migration, Transfection, Wound Healing Assay, Membrane, Expressing, Knockdown, Plasmid Preparation

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. NAC treatment (5 and 10 mM, 0-24 h) decreases N3IC protein levels in HCC1937 cells. Expression of exogenous N3ICD rescues NAC-induced inhibition of proliferation B. , migration C. , and invasion D. , and Notch3 siRNA knockdown inhibits proliferation E. , migration F. , and invasion G. in HCC1937 cells. Quantifications, sample size, statistics, and abbreviations for protein levels, proliferation, migration, and invasion assays were as described in Figure & legends.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Inhibition, Migration, Knockdown