Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: PDK1 mediates NOTCH1 -mutated head and neck squamous carcinoma vulnerability to therapeutic PI3K/mTOR inhibition

doi: 10.1158/1078-0432.CCR-18-3276

Figure Lengend Snippet: NOTCH1 loss-of-function (LOF) mutations and NOTCH1 signaling in head and neck squamous cell carcinoma (HNSCC) are correlated with sensitivity to PI3K/mTOR pathway inhibitors. A. HNSCC cell lines (n=59) were incubated with seven different PI3K/mTOR pathway inhibitors. The drugs’ IC70 and area under the curve (AUC) values in wild type cell lines were compared with those in cell lines with mutations in 33 common driver genes using a modified two-sample t-test at a false discovery rate (FDR) of 0.05. Positive and negative correlations between the presence of a mutation and drug sensitivity are indicated by blue and red data points, respectively, with larger points representing significant differences. B. The IC70 values in cell lines with wild type or mutant NOTCH1 and PIK3CA were compared using a Kruskal-Wallis test corrected for multiple comparisons by the Dunn test; p values are indicated in the figure. The dashed black lines indicate each drug’s peak plasma concentration. N.S., not significant. C. The frequency and location of mutations in NOTCH1 in HNSCC cell lines (upper gene map) are compared with those of T-acute lymphoblastic leukemia (T-ALL; lower map, below the gene) and HNSCC patients (upper map, above the gene) based on data from the Catalogue of Somatic Mutations in Cancer and The Cancer Genome Atlas (TCGA). Oncogenic T-ALL NOTCH1 mutations occur in a hotspot of mostly missense mutations within the negative regulatory heterodimerization (NRD) domain or in a second hotspot of mostly truncating mutations near the C-terminus, deleting the proline-glutamic acid-serine-threonine (PEST) domain and increasing the stabilization of activated NOTCH1 in the nucleus. In contrast, the truncating mutations in HNSCC are scattered throughout the gene but not in the PEST domain, and the missense mutations cluster in the extracellular EGF ligand-binding domains. D. Most mutations in the NOTCH1 gene in TCGA HNSCC patient samples are missense mutations outside the C-terminal NRD and PEST domains. E. From our panel of 59 HNSCC cell lines, we identified 24 harboring NOTCH1 and/or PIK3CA mutations. Orange text corresponds to nonsense mutations outside the PEST domain; red text, frameshift mutations outside the PEST domain; blue text, missense mutations outside the NRD and PEST domains; and gray text, in-frame deletions. ¥Non-canonical mutations; †suspected single-nucleotide polymorphisms excluded from NOTCH1 LOF mutants. F. The basal expression levels of NOTCH1 and NOTCH intracellular domain (NICD) proteins in NOTCH1WT and NOTCH1MUT cell lines were compared using a two-sample t-test. The correlation between the expression levels of NICD and NOTCH1 was assessed by Spearman correlation.

Article Snippet: To create the PJ34, FaDU, and MDA686LN NOTCH1 KO lines, we transfected the parental cell lines with a NOTCH1 CRISPR/Cas9 KO plasmid (sc-421930; Santa Cruz, Dallas, TX) using GenJet DNA transfection reagent (Signagen, Rockville, MD).

Techniques: Incubation, Modification, Mutagenesis, Clinical Proteomics, Concentration Assay, Ligand Binding Assay, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: PDK1 mediates NOTCH1 -mutated head and neck squamous carcinoma vulnerability to therapeutic PI3K/mTOR inhibition

doi: 10.1158/1078-0432.CCR-18-3276

Figure Lengend Snippet: PI3K/mTOR signaling inhibition induces cell death in NOTCH1MUT but not NOTCH1WT head and neck squamous cell carcinoma (HNSCC) cell lines in vitro. A. Annexin V/propidium iodide or BrDU-terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assays were used to measure apoptosis in HNSCC cells with the indicated NOTCH1 and PIK3CA mutations, which were treated with 50 nM GSK2126458 (GSK212) for 24 or 48 hours, respectively. Values are the means ± standard deviations of three independent experiments. *p<0.05, unpaired two-tailed Student t-test. B. Western blotting of cleaved PARP (Cl-PARP) and cleaved caspase 3 (Cl-Caspase 3) levels in mutant and wild type cells after GSK212 treatment for 48 hours. C. NOTCH1MUT and NOTCH1WT HNSCC cells were plated sparsely and treated with GSK212 at the indicated concentrations for 48 hours. At 14-21 days after treatment, the cells were fixed and stained, images were captured using the GelCount Tumour Colony Counter, and colony numbers and the colorimetric intensity of the colonies were determined by measuring the optical density (OD) at 570 nm. Values are the means ± standard deviations of three independent experiments. *p<0.001, unpaired two-tailed Student t-test. D. Three NOTCH1WT lines (black text and bars) and three NOTCH1MUT lines (red text and bars) were treated with increasing concentrations of GSK212 for 4 hours, and levels of PI3K/mTOR pathway proteins were measured by Western blot analysis.

Article Snippet: To create the PJ34, FaDU, and MDA686LN NOTCH1 KO lines, we transfected the parental cell lines with a NOTCH1 CRISPR/Cas9 KO plasmid (sc-421930; Santa Cruz, Dallas, TX) using GenJet DNA transfection reagent (Signagen, Rockville, MD).

Techniques: Inhibition, In Vitro, End Labeling, TUNEL Assay, Two Tailed Test, Western Blot, Mutagenesis, Staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: PDK1 mediates NOTCH1 -mutated head and neck squamous carcinoma vulnerability to therapeutic PI3K/mTOR inhibition

doi: 10.1158/1078-0432.CCR-18-3276

Figure Lengend Snippet: NOTCH1 knockout (KO) sensitizes two NOTCH1WT head and neck squamous cell carcinoma (HNSCC) cell lines to PI3K/mTOR pathway inhibition in vitro. Two NOTCH1WT HNSCC cell lines (PJ34 and UMSCC49 [UM49]), parental cell lines, and NOTCH1 KO cell lines were treated with PI3K/mTOR inhibitors. A. The CellTiter-Glo assay was used to assess the viability of cells treated for 72 hours with the indicated concentrations of GSK2126458 (GSK212). B. Western blot analysis of apoptosis markers, NOTCH1, and NOTCH intracellular domain (NICD) was performed after treatment with the indicated concentrations of GSK212 for 48 hours. C. Apoptosis in cells treated with 50 nM GSK212 or 200 nM BEZ235 for 48 hours was measured using an Annexin V/propidium iodide assay. Values are the means ± standard deviations of three independent experiments. *p<0.05, unpaired two-tailed Student t-test. D. Cells were treated with 50 nM GSK212 for 48 hours and then incubated in drug-free medium for 14-21 days. Colony areas as percentages of the control were measured using ImageJ. Data are presented as the means ± standard deviations of three independent experiments. *p<0.05, unpaired two-tailed Student t-test.

Article Snippet: To create the PJ34, FaDU, and MDA686LN NOTCH1 KO lines, we transfected the parental cell lines with a NOTCH1 CRISPR/Cas9 KO plasmid (sc-421930; Santa Cruz, Dallas, TX) using GenJet DNA transfection reagent (Signagen, Rockville, MD).

Techniques: Knock-Out, Inhibition, In Vitro, Glo Assay, Western Blot, Two Tailed Test, Incubation, Control

Journal: Cancer Research

Article Title: Hypoxia-Regulated Delta-like 1 Homologue Enhances Cancer Cell Stemness and Tumorigenicity

doi: 10.1158/0008-5472.can-09-1605

Figure Lengend Snippet: Figure 2. DLK1 maintains an undifferentiated NB phenotype. A, BE(2)C cells were differentiated with 1 μmol/L RA or 10 μmol/L BrdUrd for 5 d. DLK1, Sox2, c-kit, and CD-133 were detected by Western blot. B, BE(2)C cells were infected with lentivirus expressing shDLK-2H, shDLK-4H, or empty lentiviral vector (ctrl). Left: Infected cells were stained using antibodies against DLK1 (red) or neuron-specific β-tubulin III (green) with Hoechst 33342 as nuclear stain (blue). Magnification, ×200. Right, expression of DLK1 was analyzed by Western blot, and cells with β-tubulin III–positive neurites were enumerated from five to six random fields (columns, mean; bars, SEM; *, P < 0.0001 versus control). C, left, fluorescence-activated cell sorting (FACS)–selected BE(2)C cells expressing the full-length DLK1 (DLK-FL) or empty retroviral vector (ctrl) were cultured for 3 d with or without 1 nmol/L RA and then stained as in B. Magnification, ×200. Right, neurite-positive cells were counted from five to six random fields (columns, mean; bars, SEM; *, P = 0.0001 versus RA-treated control). D, Western blot for CD133 and Notch1 in ER cells expressing the full-length DLK1 with the empty retroviral vector as control (ctrl).

Article Snippet: Western blot was done as described previously (11) with the following antibodies: polyclonal rabbit anti-DLK1 (1:3,000; Chemicon International); anti-Sox2 (1:1,000; Chemicon International); c-kit (1:500; Zymed Laboratories); CD-133 (1:100; Abgent,); Notch1 (1:2,000), HIF-1α (1:2,000), and HIF-2α (1:1,000; Novus Biologicals); proliferating cell nuclear antigen (PCNA; 1:500; Santa Cruz Biotechnology); and phospho–extracellular signal-regulated kinase (ERK) and total ERK (1:1,000; Cell Signaling), or β-actin (1:20,000; Sigma-Aldrich).

Techniques: Western Blot, Infection, Expressing, Plasmid Preparation, Staining, Control, Fluorescence, FACS, Retroviral, Cell Culture

Journal: bioRxiv

Article Title: Prediction and validation of host cleavage targets of SARS-CoV-2 3C-like protease

doi: 10.1101/2022.01.17.476677

Figure Lengend Snippet: A) Western blot of in vitro cleavage of recombinant cadherin with purified 3CLPro. 1uM of purified 3CLPro was incubated with 2ug of recombinant CDH6 or CDH20 (C-terminal His-Tag) in a 50uL reaction for 1hr. Shown are cleavage sites within the recombinant fragment, with amino acid positions displayed for the full length proteins. Western blots showing staining against the C-terminus (His-Tag) of each protein and 3CLPro. Recombinant proteins are a mixture of glycosylated (∼90kDa) and unglycosylated (∼65kda), corresponding to cleavage fragments of ∼62kDa and 40kDa (respectively). B) Western blot of in vitro cleavage of purified human alpha thrombin (IIa). Diagram shows amino acid position of unprocessed prothrombin. Position of cleavage site shown with respect to epitope of antibody used for western blot. 1uM of purified 3CLpro was incubated with 2ug alpha thrombin overnight under reducing conditions, with or without the 3CLPro inhibitor GC376 (1uM). C) In vitro cleavage of purified recombinant NOTCH1 fragment (aa2280-2550) with a N-terminal His-Tag. Reactions were done with 1uM of purified 3CLPro for 1hr. Diagram shows position of cleavage within the NOTCH1 fragment, with amino acid positions corresponding to the full length protein. Epitope regions showed for antibody with epitope C-terminal to the cleavage site. Full length size is ∼29kDa, with N and C-terminal fragments of 4kDa and 25kDa (respectively). D) Cellular cleavage of NOTCH1. Western blots show lysates of hIPSC cardiomyocytes expressing 3CLPro or catalytically inactive C145A variant for 48h. Cleavage site position within the intracellular fragment of NOTCH1 shown, as well as epitope for antibody used in western blot. For all statistics shown, * = p ≤ 0.05 , ** p ≤ 0.01, ***p ≤ 0.001, **** = p ≤ 0.00001

Article Snippet: Recombinant proteins were purchased commercially: NOTCH1 (Origene, Cat# TP762041), CDH6 (ACROBiosystem, Cat# CA6-H5229), CDH20 (R&D, Cat# 5604-CA-050).

Techniques: Western Blot, In Vitro, Recombinant, Purification, Incubation, Staining, Expressing, Variant Assay

Journal: bioRxiv

Article Title: Prediction and validation of host cleavage targets of SARS-CoV-2 3C-like protease

doi: 10.1101/2022.01.17.476677

Figure Lengend Snippet: A) Western blots of candidate cleavage targets. 48h overexpression of 3CLPro or C145A in 293T cells. B) Western blot for the C-terminal cleavage fragment of NOTCH1 following 48h overexpression in 293T cells.

Article Snippet: Recombinant proteins were purchased commercially: NOTCH1 (Origene, Cat# TP762041), CDH6 (ACROBiosystem, Cat# CA6-H5229), CDH20 (R&D, Cat# 5604-CA-050).

Techniques: Western Blot, Over Expression

Journal: The Journal of Clinical Investigation

Article Title: DLL4 + neutrophils promote Notch1-mediated endothelial PANoptosis to exacerbate acute lung injury in sepsis

doi: 10.1172/JCI194310

Figure Lengend Snippet: ( A ) Computational modeling for the protein-protein interaction analysis between Notch1 extracellular domain and DLL4. Notch1 is shown in green, DLL4 in blue, and the interaction between Notch1 and DLL4 in purple. ( B ) Computational modeling was used to design Notch1-DLL4 inhibitor (NDI) derived from the Notch1 sequence that binds to DLL4. DLL4 is shown in blue, NDI in red. ( C and D ) BIAcore analysis was conducted to determine the binding affinity ( K D value) of DLL4 to Notch1 and the inhibitory effect of NDI on this interaction. Wild-type PVECs (0.5 × 10 6 ) were cocultured with sorted DLL4 + neutrophils (0.5 × 10 6 ) or DLL4 – neutrophils (0.5 × 10 6 ), then treated with either 10 μM scramble or NDI. PVECs (0.5 × 10 6 ) were first treated with Notch1-knockdown plasmid or its negative control for 24 hours. These PVECs were then cocultured with sorted DLL4 + neutrophils (0.5 × 10 6 ). ( E ) After 16 hours, total RNA was extracted and ZBP1 expression was analyzed by RT-PCR. Experiments were performed at least 3 times, and all data were analyzed. Data are expressed as means ± SEM and were analyzed using 1-way ANOVA. n = 8–15 per group. ( F – I ) Western blot analysis was performed to measure protein levels of ZBP1, c-GSDMD, t-GSDMD, c-caspase-3, t-caspase-3, p-MLKL, and MLKL in PVECs. Pyroptosis, apoptosis, and necroptosis were quantified as c/t GSDMD, c/t caspase-3, and p-MLKL/MLKL. n = 6–14 per group. Data are representative of 3 independent experiments. Data are expressed as means ± SEM and were analyzed using 1-way ANOVA. * P < 0.05 vs. PBS. # P < 0.05 vs. DLL4 + neutrophils/scramble. Con, Notch1-knockdown plasmid negative control–treated PVECs; Ko, Notch1-knockdown plasmid–treated PVECs.

Article Snippet: PVECs were transfected with a double-nickase plasmid targeting Notch1 using the UltraCruz transfection reagent (Santa Cruz Biotechnology), according to the manufacturer’s protocol.

Techniques: Derivative Assay, Sequencing, Binding Assay, Knockdown, Plasmid Preparation, Negative Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: DLL4 + neutrophils promote Notch1-mediated endothelial PANoptosis to exacerbate acute lung injury in sepsis

doi: 10.1172/JCI194310

Figure Lengend Snippet: DLL4 + neutrophils induce pulmonary endothelial cell PANoptosis via the Notch1-DLL4 pathway in sepsis. eCIRP induces neutrophils to express DLL4, and these DLL4 + neutrophils interact with PVECs through the Notch1-DLL4 pathway. This interaction triggers endothelial PANoptosis, driving inflammation and ALI in sepsis. NDI effectively inhibits the Notch1-DLL4 interaction, reducing endothelial PANoptosis and ALI, thereby improving survival outcomes in sepsis. The schema was created in BioRender (Murao A, 2025, https://BioRender.com/y914a2i ).

Article Snippet: PVECs were transfected with a double-nickase plasmid targeting Notch1 using the UltraCruz transfection reagent (Santa Cruz Biotechnology), according to the manufacturer’s protocol.

Techniques: