Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Ectopic expression of ICN1 mimics changes induced by JAG1 stimulation. A. Infection with ICN1 but not empty vector (MigR1) strongly inhibited protein expression of AXL and α-CATULIN. Widely used ICN1 cDNA product which begins several amino acids downstream of the native cleavage site is recognized by antibodies to the C-terminal region of NOTCH1 but not antibodies specific to the cleavage site. B. Expression of ICN1 in NOTCH1 mutant UMSCC22A produced the same morphological changes observed earlier upon expression of NFL1 and growth on JAG1. C. Expression of ICN1 in NOTCH1 WT 183 produced the same morphological changes observed earlier with growth on JAG1. D. Expression of ICN1 but not MigR1 control in NOTCH1 WT PJ34 produced the same morphological changes observed earlier with growth on JAG1. E. ICN1 expression triggered decreased protein expression of AXL, α-CATULIN, and ITGA3 in 183 cells.

Article Snippet: For NOTCH activation experiments, tissue culture wells were pre-coated overnight at room temperature (RT) with Protein G (Prospec, East Brunswick, NJ) at 50 ug/ml, washed twice in phosphate buffered saline (PBS), blocked in 1% BSA/PBS for 2 hours at RT, washed three times with PBS, coated with either human recombinant chimeric Jag1 fused to an FC fragment (R&D Systems, Minneapolis, MN) or control purified IgG FC protein (Jackson ImmunoResearch, West Grove, PA) at 2 ug/ml in 0.1% BSA/PBS for 3 hours RT, stored overnight at 4 0 C, and washed three times immediately before use.

Techniques: Expressing, Infection, Plasmid Preparation, Mutagenesis, Produced, Control

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Activation of NOTCH1 fails to increase surface CD133+ but increases expression of SOX2 in NOTCH1 mutant (UMSCC22A-iICN1), or NOTCH1 WT cell lines (FaDu-iICN1 and PJ34-iICN1). A. Cells were incubated for 48 h with or without DOX at 200 ng/ml (UMSCC22A), 300 ng/ml (FaDu), or 1000 ng/ml (PJ34) before staining with antibody to surface CD133 by flow cytometry. Fluorescent intensity histograms are shown for control biological replicates without DOX (red and light blue traces) or after DOX treatment (green and orange traces). B. Statistical comparison of CD133 mean fluorescence intensity (MFI). C. Western blot analysis of SOX2 protein expression in similarly treated cells.

Article Snippet: For NOTCH activation experiments, tissue culture wells were pre-coated overnight at room temperature (RT) with Protein G (Prospec, East Brunswick, NJ) at 50 ug/ml, washed twice in phosphate buffered saline (PBS), blocked in 1% BSA/PBS for 2 hours at RT, washed three times with PBS, coated with either human recombinant chimeric Jag1 fused to an FC fragment (R&D Systems, Minneapolis, MN) or control purified IgG FC protein (Jackson ImmunoResearch, West Grove, PA) at 2 ug/ml in 0.1% BSA/PBS for 3 hours RT, stored overnight at 4 0 C, and washed three times immediately before use.

Techniques: Activation Assay, Expressing, Mutagenesis, Incubation, Staining, Flow Cytometry, Control, Comparison, Fluorescence, Western Blot

Journal: bioRxiv

Article Title: P66 is a bacterial mimic of CD47 that binds the anti-phagocytic receptor SIRPα and facilitates macrophage evasion by Borrelia burgdorferi

doi: 10.1101/2024.04.29.591704

Figure Lengend Snippet: The surface protein P66 binds to a high-affinity CD47 binding reagent and the CD47 receptor SIRPα. A. B. burgdorferi stain for the high-affinity CD47 blocking reagent CV1-G4 by flow cytometry analysis. **** p-value < 0.0001, df = 4, N = 4; Unpaired 2-tailed t-test. B. B. burgdorferi was cultured under conditions to stain either positively or negatively for CV1-G4 as confirmed by flow cytometry. Bacteria were lysed under non-denaturing conditions, and lysate was subjected to enrichment with CV1-G4 or IgG4 prior to SDS-PAGE. Gel bands of interest were excised and subjected to in-gel trypsin digestion followed by mass spectrometry analysis. Arrow denotes putative P66 band. C. Data filtration parameters identified P66 as a putative CV1-G4 binding protein. D. Recombinant his-tagged P66 is enriched by CV1-G4 or SIRPα but not isotype control as determined by in vitro binding assay. A representative blot is shown as is quantification from 4 replicates. ** p-value = 0.0065, N = 4, df = 3. Paired two-tailed t-test; *** p-value < 0.0008, N = 4, df = 3;. Error bars calculated as SEM.

Article Snippet: 10 9 E. coli expressing P66, or GFP as a negative control, were suspended in 200 μL of a 125 nM solution of recombinant human SIRPα-Fc protein (R&D Systems), the cognate isotype control recombinant human IgG1 protein (Sino Biological), or SIRPα-His.

Techniques: Binding Assay, Staining, Blocking Assay, Flow Cytometry, Cell Culture, Bacteria, SDS Page, Mass Spectrometry, Filtration, Recombinant, In Vitro, Two Tailed Test

Journal: bioRxiv

Article Title: P66 is a bacterial mimic of CD47 that binds the anti-phagocytic receptor SIRPα and facilitates macrophage evasion by Borrelia burgdorferi

doi: 10.1101/2024.04.29.591704

Figure Lengend Snippet: P66 is necessary and sufficient for binding to a CD47 affinity reagent and SIRPα. A. Wild-type (WT), p66 deficient (Δ p66 ) B. burgdorferi , Δ p66 B. burgdorferi reconstituted with WT p66 , Δ p66 B. burgdorferi reconstituted with the p66 D184A D186A mutant, or Δ p66 B. burgdorferi reconstituted with the p66 Δ 181-187 loop deletion mutant were stained with CV1-G4, IgG4 or MIAP410 and analyzed by flow cytometry. B. Predicted structure of P66 by Alphafold2 with mutated residues or loop deletions highlighted in orange. C. P66-His- or GFP-expressing E. coli were incubated with soluble SIRPa-Fc or IgG1 isotype control, prior to Western blot analysis. D. Soluble SIRPa-Fc, SIRPa-His or IgG1 isotype control was incubated with E. coli expressing P66-His or GFP prior to Western blot analysis.

Article Snippet: 10 9 E. coli expressing P66, or GFP as a negative control, were suspended in 200 μL of a 125 nM solution of recombinant human SIRPα-Fc protein (R&D Systems), the cognate isotype control recombinant human IgG1 protein (Sino Biological), or SIRPα-His.

Techniques: Binding Assay, Mutagenesis, Staining, Flow Cytometry, Expressing, Incubation, Western Blot