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: Journal of Inflammation Research

Article Title: Aryl Hydrocarbon Receptor is Essential in the Control of Lung Club Cell Homeostasis

doi: 10.2147/JIR.S284800

Figure Lengend Snippet: Expression levels of Notch1 signaling molecules are reduced in club cell-specific AhR-null mice. Lung sections were stained for Notch1 (red; (A)) or Hes5 (red; (B)) and CC10 (green) and DAPI (blue)), and the quantification data were showed in ( C ) and (D) (mean ± SEM scores were obtained from 3 animals), respectively. **P < 0.01, ***P < 0.001 ( t tests).

Article Snippet: Western blotting and immunohistochemical (fluorescence) staining analyses were performed as described previously , with the following primary antibodies: actin monoclonal (1:5000 dilution; Sigma–Aldrich), FITC-conjugated anti-goat IgG, rhodamine-conjugated anti-rabbit IgG, alkaline phosphatase-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories), AhR and CC10 goat polyclonal antibodies (Santa Cruz Biotechnology), Notch1 and Hes5 rabbit polyclonal antibodies (cell signaling technology), AhR monoclonal antibody (Enzo life Sciences), and GFP rabbit polyclonal antibody (Abcam).

Techniques: Expressing, Staining

Journal: Journal of Inflammation Research

Article Title: Aryl Hydrocarbon Receptor is Essential in the Control of Lung Club Cell Homeostasis

doi: 10.2147/JIR.S284800

Figure Lengend Snippet: AhR is required for Notch1 mRNA and protein expression in Lung epithelial NL-20 cells; ( A ) NL-20 cells were treated with the AhR ligand Indeno[1,2,3-cd]pyrene (IP) at 1μM, and were examined in Western blotting analyses. Notch1 (NTM) is transmembrane/intracellular protein. ( B ) Doxycycline mediated ectopic AhR expression induces Notch1 in NL-20 cells. ( C ) IP-treated AhR-null cells showed lower expression levels of NOTCH1 and the downstream gene HES5 in NL-20 cells. ( D ) IP-treated (1μM) AhR-null cells had attenuated Notch1 (NTM) expression in NL-20 cells. ( E ) Serial deletion constructs of Notch1 promoters for promoter-reporter assays; ( F ) A549 cells were transfected with a series of deletion constructs of the Notch1 promoter and luciferase activities were determined. ( G ) A549 cells with ectopic AhR-GFP expression were analyzed in chromatin immunoprecipitation assays with IgG and GFP antibodies; *P < 0.05, **P < 0.01, ****P < 0.0001 ( t tests or One-Way ANOVA).

Article Snippet: Western blotting and immunohistochemical (fluorescence) staining analyses were performed as described previously , with the following primary antibodies: actin monoclonal (1:5000 dilution; Sigma–Aldrich), FITC-conjugated anti-goat IgG, rhodamine-conjugated anti-rabbit IgG, alkaline phosphatase-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories), AhR and CC10 goat polyclonal antibodies (Santa Cruz Biotechnology), Notch1 and Hes5 rabbit polyclonal antibodies (cell signaling technology), AhR monoclonal antibody (Enzo life Sciences), and GFP rabbit polyclonal antibody (Abcam).

Techniques: Expressing, Western Blot, Construct, Transfection, Luciferase, Chromatin Immunoprecipitation

Journal: Cell

Article Title: Dynamic Ligand Discrimination in the Notch Signaling Pathway

doi: 10.1016/j.cell.2018.01.002

Figure Lengend Snippet: (A) C2C12 cells were engineered to expressed Notch1 receptors lacking the extracellular domain (N1DECD, green). This receptor is inactive in the presence of the γ-secretase inhibitor DAPT (red), but constitutively active when DAPT concentration is reduced in the culture medium. (B) Comparison of transcript levels in C2C12-N1ΔECD cells at 1 hr or 6 hr after DAPT removal. The blue line represents equal expression at 1 hr and 6 hr, and the gray lines represent 5-fold changes in either direction. Circled genes are putative direct Notch targets. The blue circle highlights target genes that are upregulated >5-fold at 1 hr but not 6 hr, while red circles indicate target genes that are upregulated >5-fold only after 6 hr. See also and . (C) qPCR time course measurement of Hes1 (blue), Hey1 (orange), and HeyL (yellow) mRNA levels following complete DAPT removal at t = 0 hr. (D) Duration dependence of Hes1 (blue) and Hey1 (orange) response to DAPT removal for 5 min, 15 min, or 30 min followed by replenishment (“Pulse”), or no replenishment until the 1 hr or 4 hr measurement (“Sustained”). Error bars represent SEM calculated from duplicate experiments (n = 2). See also Figure S4 .

Article Snippet: Cells were then incubated with recombinant mouse Notch1 ext -Fc protein in binding solution (blocking solution containing 100 ug/ml CaCl 2 , R&D Systems) for 45 min at RT.

Techniques: Concentration Assay, Comparison, Expressing

Journal: Cell

Article Title: Dynamic Ligand Discrimination in the Notch Signaling Pathway

doi: 10.1016/j.cell.2018.01.002

Figure Lengend Snippet: (A) Both Dll1 (blue) and Dll4 (red) activate the Notch1 receptor (green) to induce proteolytic release of the Notch intracellular domain (NICD), but are used in different biological contexts (blue and red boxes, bottom). The released NICD translocates to the nucleus and, in complex with CSL/RBPjκ (yellow), activates Notch target genes (white). (B) Left: Engineered CHO-K1 “sender” cell lines contain stably integrated constructs expressing Dll1 (blue) or Dll4 (red), each with a co-translational (T2A, brown) H2B-mCh readout (purple), from a 4epi-Tetracycline (4epi-Tc) inducible promoter. Right: “Receiver” cells stably express a chimeric receptor combining the Notch1 extracellular domain (Notch1ECD) with a Gal4 transcription factor (orange), which can activate a stably integrated fluorescent H2B-3xCitrine reporter gene (chartreuse). (C) Left (schematics): A minority of receiver cells (green) are co-cultured with an excess of either Dll1 (blue) or Dll4 (red) sender cells. Right: Filmstrips showing representative sustained (top, Dll4 senders) or pulsatile (bottom, Dll1 senders) response of a single receiver cell (center, automatically segmented nucleus outlined in white). Grey channel shows DIC images of cells, while the rate of increase in Citrine fluorescence, scaled to 25%–75% of its total range, is indicated using green pseudo-coloring. See also and . (D) Left: Representative traces showing total nuclear Citrine fluorescence levels (top) or corresponding derivatives of the total Citrine ( d Citrine/ d t), i.e., promoter activity (bottom), in individual receiver cells activated by Dll4. Right: Average values of total fluorescence (top) and promoter activity (bottom) in receiver cells activated by Dll4. Solid traces represent medians, lighter shades indicate SEM, and gray shading indicates SD. n, number of traces included in the alignment. See for alignment and normalization procedure. (E) Left: Corresponding plots (as in D) showing total nuclear Citrine fluorescence levels (top) and promoter activity (bottom) in individual receiver cells in co-culture with Dll1. Right: Average values of total fluorescence (top) and promoter activity (bottom) in receiver cells activated by Dll1. The percentage value (60%) in the plots on right indicates the fraction of receiver traces included in the alignment ( STAR Methods , see also ). (F) 95 th percentile of (absolute, non-normalized) promoter activity values between 0 and 7.5 hr (after alignment) in the traces included in (D) and (E). This time window is chosen to simultaneously estimate the promoter activity at the peak of Dll1 pulses and at steady-state levels of Dll4 signaling. Solid horizontal lines represent medians, while the boxes delineate 25 th –75 th percentile values. p value calculated by two-sided Kolmogorov-Smirnov (K-S) test. See also Figures S1 and S2 .

Article Snippet: Cells were then incubated with recombinant mouse Notch1 ext -Fc protein in binding solution (blocking solution containing 100 ug/ml CaCl 2 , R&D Systems) for 45 min at RT.

Techniques: Stable Transfection, Construct, Expressing, Cell Culture, Fluorescence, Activity Assay, Co-Culture Assay

Journal: Cell

Article Title: Dynamic Ligand Discrimination in the Notch Signaling Pathway

doi: 10.1016/j.cell.2018.01.002

Figure Lengend Snippet: (A) Developing chick embryo (dorsal view schematic). Dll1 (blue cells in 3) is expressed in a fraction of neural crest cells (gray, see 2, 3). These cells activate Notch1-expressing Pax7 + progenitor cells in the dorsomedial lip (DML, magenta) of the somite. When activated, these progenitor cells (green, 3) upregulate Hes1 and the muscle regulatory gene MyoD1. (B–D) Representative images showing effects of Dll1 or Dll4 electroporation into the neural crest, on Hes1, Hey1, and MyoD1 expression in the DML. White arrows indicate the somites on the electroporated side. The dotted lines indicate the DMLs of somites or the central line of the neural tube. (B) Top: Dll1-T2A-EGFP (i, blue), electroporated into the left side of the neural tube, is expressed in the neural tube and neural crest, resulting in upregulation of Hes1 (ii, red) and MyoD1 (iii, green) in the somites on the electroporated (left) side compared to the right side, which serves as negative control. Bottom: When Dll4-T2A-EGFP (iv, blue) is electroporated, Hey1 (v, red) is upregulated on the electroporated side, and MyoD1 (vi, green) expression is decreased. (C) Dll1-T2A-EGFP (blue, left) electroporation does not affect expression of Hey1 (red, right) in adjacent somites. (D) Dll4-T2A-EGFP (blue, left) electroporation increases expression of Hes1 (red, right) in adjacent somites. See also and .

Article Snippet: Cells were then incubated with recombinant mouse Notch1 ext -Fc protein in binding solution (blocking solution containing 100 ug/ml CaCl 2 , R&D Systems) for 45 min at RT.

Techniques: Expressing, Electroporation, Negative Control

Journal: Cell

Article Title: Dynamic Ligand Discrimination in the Notch Signaling Pathway

doi: 10.1016/j.cell.2018.01.002

Figure Lengend Snippet: (A and B) Dll4 ECD -Dll1 ICD and Dll1 ECD -Dll4 ICD were constructed by exchanging the intracellular domain (ICD) of Dll4 with that of Dll1. (A) Median response profiles in activated receiver cells co-cultured with Dll4 sender cells (red, top left) or Dll4 ECD -Dll1 ICD sender cells (magenta, right) under excess receiver conditions (as in ). Solid traces represent medians, lighter colored regions represent SEM, and gray shading represents SD. n, number of cell traces included in the alignment. See for alignment and normalization procedures. Bottom left: 95 th percentile of (absolute, non-normalized) promoter activity values between 0 and 7.5 hr (after alignment) in individual traces included in the averaging. Solid horizontal lines represent medians, while the boxes delineate 25 th –75 th percentile values. p value calculated by two-sided K-S test. (B) Corresponding response profiles (right, top left) and amplitudes (bottom left) in activated receiver cells co-cultured with Dll1 sender cells (blue) or Dll1 ECD- Dll4 ICD sender cells (purple) under excess sender conditions. (C) Representative images of “excess sender” co-cultures of receiver cells (R) expressing full-length Notch1 and sender cells (S) expressing either Dll4 ECD -Dll1 ICD (left) or Dll4 (Dll4 ECD -Dll4 ICD , right), immunostained for Notch1ECD. Examples of dispersed, low intensity staining or higher-intensity puncta are indicated by the white circles. (D) Left: Median values of number of puncta detected (see ) in Dll1 ICD (blue) or Dll4 ICD (red) sender cells neighboring receiver cells. Right: Median values of the (background subtracted) mean pixel intensity of dispersed signal (see ) within Dll1 ICD (blue) or Dll4 ICD (red) sender cells that neighbor receiver cells. Error bars represent SEM. p value calculated using the two-sided K-S test. (E) Schematic: Proposed differences in the abilities of ligands containing the Dll1 (blue) and Dll4 (red) ICDs to initiate transendocytosis in different clustering states. See also Figure S6 .

Article Snippet: Cells were then incubated with recombinant mouse Notch1 ext -Fc protein in binding solution (blocking solution containing 100 ug/ml CaCl 2 , R&D Systems) for 45 min at RT.

Techniques: Construct, Cell Culture, Activity Assay, Expressing, Staining

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Dietary nitrate inhibits the Notch pathway overactivated by ethanol in SD rats. (A) Bubble plot showing the top 20 significantly enriched KEGG pathways. The abscissa is the ratio of the number of differential genes annotated to the KEGG pathway to the total number of differential genes, and the ordinate is the KEGG pathway. (B) Representative immunoblotting band of Notch1, Nicd, and Rbpj protein in gastric mucosal tissue. (C–E) Analyses of immunoblotting band gray value in (B). (F) GSEA analysis of Notch signaling pathway between ulcer and control groups. (G) GSEA analysis of Notch signaling pathway between ulcer + Nit and ulcer groups. Quantitative data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. KEGG, Kyoto Encyclopedia of Genes and Genomes; Nicd, intracellular structural domain; Rbpj, recombination signal binding protein for immunoglobulin kappa J region; GSEA, gene set enrichment analysis; RNA seq, RNA sequencing; SD,D standard deviation.

Article Snippet: Following incubation with primary NICD (NBP1‐48289; Novus Biologicals, USA) and RBPJ (720219; Thermo, USA) antibodies, a pair of Duolink PLA probes—anti‐mouse MINUS (DUO92004; Merck) and anti‐rabbit PLUS (DUO92002; Merck)—were applied for 60 min. Ligase was added for 30 min followed by amplification of signal for 100 min using polymerase.

Techniques: Western Blot, Control, Binding Assay, RNA Sequencing, Standard Deviation

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Nitrate functions by Notch pathway inhibition positively transcripting TFF2 in vitro. (A) Diagram of the wild‐type and mutant sequences for the three predicted RBPJ binding sites within the 251 bp TFF2 promoter probes. (B) Representative electrophoretic mobility shift assay (EMSA) autoradiograph. Hot probe is the biotin‐labeled wild‐type oligonucleotides of the truncated TFF2 promoter containing the binding motif; Mutant probe is the labeled oligonucleotides sequence with nucleotides mutated. The cold probe is nonlabeled competitive wild‐type probes (100 and 50 that of the concentrations). The shifted bands are indicated by arrows, which suggested the formation of DNA–protein complexes (lane 2, 3, 7). The super shifted bands indicated the formation of DNA–protein–antibody complexes (lane 3, 7). “+” and “−” represent presence and absence, respectively. (C) Relative TFF2 promoter (Full, Mut1, Mut2, and Mut3) luciferase activity was detected by DLR assays in RBPJ overexpressed and normal‐expressed GES‐1 cells. (D) A schematic diagram showing the location of RBPJ putative binding regions on the TFF2 promoter. (E) RT‐qPCR analysis of TFF2 binding site expression of GES‐1 cells. Target site expression in RBPJ‐treated groups was normalized to IgG negative control groups and expressed as fold change relative to the IgG groups. (F) IF staining of NICD (pink) and DAPI (blue). Scale bar = 50 µm. (G) IF analysis of MFI of nuclear NICD in positive cells. (H) Representative immunoblotting band of Notch signaling pathway in GES‐1 cells. (I–K) Analyses of immunoblotting band gray value of (H). (L) RT‐qPCR analysis of TFF2 mRNA expression of DMSO/DAPT treated GES‐1 cells. Target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the DMSO vehicle group. (M) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD deprived GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector + DMSO group. (N) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD‐RBPJ overexpressed GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector1 + vector2 group. (O) Representative image of PLA of NICD–RBPJ proximity. Each red dot represents a positive signal of NICD–RBPJ interaction and nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. Quantitative data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns denotes no significance. Nit, nitrate; EtOH, ethanol; GES‐1, human gastric epithelial; NICD, intracellular structural domain; RBPJ, recombination signal binding protein for immunoglobulin kappa J region; EMSA, electrophoretic mobility shift assay; TFF2, trefoil factor 2; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; Yhhu‐3792, N2‐(4‐isopropylphenyl)‐5‐(3‐methoxyphenoxy) quinazoline‐2,4‐diamine; Luc, luciferase; SD, standard deviation; DLR, dual‐luciferase report; PLA, proximity ligation assay.

Article Snippet: Following incubation with primary NICD (NBP1‐48289; Novus Biologicals, USA) and RBPJ (720219; Thermo, USA) antibodies, a pair of Duolink PLA probes—anti‐mouse MINUS (DUO92004; Merck) and anti‐rabbit PLUS (DUO92002; Merck)—were applied for 60 min. Ligase was added for 30 min followed by amplification of signal for 100 min using polymerase.

Techniques: Inhibition, In Vitro, Mutagenesis, Binding Assay, Electrophoretic Mobility Shift Assay, Autoradiography, Labeling, Sequencing, Luciferase, Activity Assay, Quantitative RT-PCR, Expressing, Negative Control, Staining, Western Blot, Targeted Gene Expression, Plasmid Preparation, Standard Deviation, Proximity Ligation Assay