Journal: Cell

Article Title: Neutrophil elastase selectively kills cancer cells and attenuates tumorigenesis

doi: 10.1016/j.cell.2021.04.016

Figure Lengend Snippet: (A) Heatmap of ELANE’s (3μg/mL) effects on survival, stress, and apoptosis pathways in cancer and non-cancer cells. See also Figure S3 for raw data and quantification. (B) Effect of ELANE (3μg/mL) on CD95 cleavage in cancer cells assessed by immunoblotting using anti-C-terminal CD95 antibody. *, CD95 fragment. (C) Cleavage of the N-terminal or C-terminal domains of recombinant human CD95 by ELANE was assessed by SDS-PAGE and Coomassie blue staining. Veh = PBS. (D) Bands from (C) were analyzed by mass spectrometry to identify ELANE cleavage sites (ie. non-tryptic peptides). (E) Schematic of ELANE cleavage sites in human CD95. Heatmap shows overlap to ELANE’s sequence specificity (https://www.ebi.ac.uk/merops). (F) Effect of Dynasore (60μM, 30min), control siRNA (siCTRL) or NRP1 siRNA (siNRP1) on MDA-MB-231 and A549 cell viability (calcein) following treatment with ELANE (1.2μg/mL, 4h); n=6/group. ELANE uptake was quantified by catalytic activity in cell lysates 30min post ELANE treatment and presented below. (G) Cells transduced with various human CD95 constructs (with dTomato) were treated with ELANE (3μg/mL, 30min). Apoptosis was quantified by ANXA5 staining on dTomato+ and dTomato- populations. Flow cytometric analysis of CD95 expression levels in dTomato+ versus dTomato- cells 48h post-transduction (fold-change in parentheses). n=2/group. (H) CRISPR knockdown (KD) of CD95 in MDA-MB-231 and A549 cells was measured by flow cytometry (top) and immunoblotting (bottom). (I) Effects of ELANE (6h) on parental or CD95 KD colony viability (calcein-AM); n=6/group (J) Cells were transduced to express various human CD95 constructs or GFP (top). Cell viability was determined by calcein-AM; n=10/group (bottom). See also Figures S5A–B. (K) Heatmap of the effects DDELANE expression on survival, stress, and apoptosis pathways in cancer and non-cancer cells. See also Figures S5 for raw data and quantification. *, p<0.05 Student’s t-test, data are mean ± SEM.

Article Snippet: Cancer cells were transfected with control siRNA (sc-37007) or NRP1 siRNA (sc-36038) using siRNA transfection reagents in siRNA transfection medium (sc-36868) according to the manufacturer’s protocol (Santa Cruz Biotechnology).

Techniques: Western Blot, Recombinant, SDS Page, Staining, Mass Spectrometry, Sequencing, Control, Activity Assay, Transduction, Construct, Expressing, CRISPR, Knockdown, Flow Cytometry

Journal: Cell

Article Title: Neutrophil elastase selectively kills cancer cells and attenuates tumorigenesis

doi: 10.1016/j.cell.2021.04.016

Figure Lengend Snippet: (A) Effect of FLAG-DDELANE transduction on cancer and non-cancer cell viability (calcein-AM) 56h post-transduction; n=6/group (A). (B) Proteomic quantification of H1 isoform levels in anti-FLAG co-immunoprecipitations (36h post-transduction). See also Table S4. (C) Immunofluorescence of H1.0 (red) in MDA-MB-231 and MCF10A cells. Blue = DAPI. (D-E) Proximity ligation assay (PLA) of CD95 and H1.0 in MDA-MB-231 and MCF10A following treatment with ELANE (1.5μg/mL). Representative images are shown at low (D) and high (E) magnification. (F) Quantification of PLA signals in whole cells (left) and mitochondria (right). n=100 cells/group. (G) Immunoblotting of histone H1 isoforms in cancer and non-cancer cells. (H) Effects of control (siCTRL), H1.0 (siH1.0), or H1.2 (siH1.2) siRNA on MDA-MB-231 cell viability (calcein-AM) following treatment with ELANE (1.5μg/mL, 6h); n=6/group. Inset: Immunoblots confirm H1.0 and H1.2 knockdown. (I) Effects Alexa Fluor™488-labeled histone H1.0 pre-treatment (3h) on MCF10A cell viability (calcein-AM) following ELANE treatment (1.5μg/mL, 18h). n=6/group. Representative images of H1.0 uptake (20μg/mL) at various time points (right). *, p<0.05 Student’s t-test, data are mean ± SEM. Scale bars = 20μm.

Article Snippet: Cancer cells were transfected with control siRNA (sc-37007) or NRP1 siRNA (sc-36038) using siRNA transfection reagents in siRNA transfection medium (sc-36868) according to the manufacturer’s protocol (Santa Cruz Biotechnology).

Techniques: Transduction, Immunofluorescence, Proximity Ligation Assay, Western Blot, Control, Knockdown, Labeling

Journal: Cell

Article Title: Neutrophil elastase selectively kills cancer cells and attenuates tumorigenesis

doi: 10.1016/j.cell.2021.04.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cancer cells were transfected with control siRNA (sc-37007) or NRP1 siRNA (sc-36038) using siRNA transfection reagents in siRNA transfection medium (sc-36868) according to the manufacturer’s protocol (Santa Cruz Biotechnology).

Techniques: Control, Virus, Recombinant, Western Blot, Giemsa Stain, Protease Inhibitor, Caspase-Glo Assay, Plasmid Preparation, Labeling, In Situ, Proximity Ligation Assay, Transfection, Magnetic Beads, Software, Cell Culture, Membrane

Journal: Cell

Article Title: Neutrophil elastase selectively kills cancer cells and attenuates tumorigenesis

doi: 10.1016/j.cell.2021.04.016

Figure Lengend Snippet: (A) Heatmap of ELANE’s (3μg/mL) effects on survival, stress, and apoptosis pathways in cancer and non-cancer cells. See also Figure S3 for raw data and quantification. (B) Effect of ELANE (3μg/mL) on CD95 cleavage in cancer cells assessed by immunoblotting using anti-C-terminal CD95 antibody. *, CD95 fragment. (C) Cleavage of the N-terminal or C-terminal domains of recombinant human CD95 by ELANE was assessed by SDS-PAGE and Coomassie blue staining. Veh = PBS. (D) Bands from (C) were analyzed by mass spectrometry to identify ELANE cleavage sites (ie. non-tryptic peptides). (E) Schematic of ELANE cleavage sites in human CD95. Heatmap shows overlap to ELANE’s sequence specificity (https://www.ebi.ac.uk/merops). (F) Effect of Dynasore (60μM, 30min), control siRNA (siCTRL) or NRP1 siRNA (siNRP1) on MDA-MB-231 and A549 cell viability (calcein) following treatment with ELANE (1.2μg/mL, 4h); n=6/group. ELANE uptake was quantified by catalytic activity in cell lysates 30min post ELANE treatment and presented below. (G) Cells transduced with various human CD95 constructs (with dTomato) were treated with ELANE (3μg/mL, 30min). Apoptosis was quantified by ANXA5 staining on dTomato+ and dTomato- populations. Flow cytometric analysis of CD95 expression levels in dTomato+ versus dTomato- cells 48h post-transduction (fold-change in parentheses). n=2/group. (H) CRISPR knockdown (KD) of CD95 in MDA-MB-231 and A549 cells was measured by flow cytometry (top) and immunoblotting (bottom). (I) Effects of ELANE (6h) on parental or CD95 KD colony viability (calcein-AM); n=6/group (J) Cells were transduced to express various human CD95 constructs or GFP (top). Cell viability was determined by calcein-AM; n=10/group (bottom). See also Figures S5A–B. (K) Heatmap of the effects DDELANE expression on survival, stress, and apoptosis pathways in cancer and non-cancer cells. See also Figures S5 for raw data and quantification. *, p<0.05 Student’s t-test, data are mean ± SEM.

Article Snippet: siNRP1 , Santa Cruz Biotechnology , Cat# sc-36038.

Techniques: Western Blot, Recombinant, SDS Page, Staining, Mass Spectrometry, Sequencing, Control, Activity Assay, Transduction, Construct, Expressing, CRISPR, Knockdown, Flow Cytometry

Journal: Cell

Article Title: Neutrophil elastase selectively kills cancer cells and attenuates tumorigenesis

doi: 10.1016/j.cell.2021.04.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: siNRP1 , Santa Cruz Biotechnology , Cat# sc-36038.

Techniques: Control, Virus, Recombinant, Western Blot, Giemsa Stain, Protease Inhibitor, Caspase-Glo Assay, Plasmid Preparation, Labeling, In Situ, Proximity Ligation Assay, Transfection, Magnetic Beads, Software, Cell Culture, Membrane