Journal: PLoS ONE

Article Title: Nuclear Legumain Activity in Colorectal Cancer

doi: 10.1371/journal.pone.0052980

Figure Lengend Snippet: (A) Immunoblots of legumain in lysosomal (L) and nuclear (N) fractions enriched from HCT116 and SW620 cells using density gradient centrifugation. All lanes were loaded with 15 µg total protein from each fraction. Purity controls of the subcellular fractions were assessed by staining for ARSB (soluble lysosomal protein) and SP1 (nuclear transcription factor). (B) Immunoblots of legumain (top panels), cystatin E/M (second panels) and cathepsin L (third panels) in enriched subcellular compartments isolated from HCT116 and SW620 cells using a commercial kit: Cytosol (C), membranes/lysosomes (M/L), nuclear soluble (NS), nuclear chromatin bound (NC), total lysate (TL) and conditioned media (CM). All lanes were loaded with 15 µg total protein from each fraction, except conditioned media where proteins precipitated from 1 ml was loaded. Purity controls of the different subcellular fractions were assessed by staining for α-tubulin (cytosolic protein), ARSB (soluble lysosomal protein), lamp-2 (lysosome membrane-associated protein), SP1 (nuclear transcription factor) and histone H3 (nuclear chromatin bound protein). Uncut immunoblots of legumain and cathepsin L .

Article Snippet: Control slides were prepared using buffer without substrate, the epoxy inhibitor E64 (Sigma) at a final concentration of 1 μM or human recombinant cystatin E/M (R&D Systems, 1286-PI) at a final concentration of 0.1 μM.

Techniques: Western Blot, Gradient Centrifugation, Staining, Isolation, Membrane

Journal: PLoS ONE

Article Title: Nuclear Legumain Activity in Colorectal Cancer

doi: 10.1371/journal.pone.0052980

Figure Lengend Snippet: (A) In situ proteolytic activity (green) captured by fluorescence microscopy imaging of adjacent cryosections from a HCT116 subcutaneous xenograft incubated with (top left) and without (top right) legumain substrate, legumain substrate and E64 (lower left) or legumain substrate and recombinant cystatin E/M (lower right), demonstrating the specificity of the synthetic peptide Suc-Ala-Ala-Asn-NHNapOME utilized as legumain substrate. All pictures were taken using true colors, after the same incubation time and with identical microscope and camera settings. Scale bar represents 100 µm. Subcutaneous xenografts with SW620 cells . (B) Subcellular localization of active legumain (green) in HCT116 cells (made from cryosections after mounting in OCT-medium) with nuclei stained by DAPI (red) and analyzed by confocal laser scanning microscopy. This showed granulated activity inside (yellow arrow) and outside (gray arrow) of the nucleus. Localization in the nucleus was confirmed by co-localization (white) of legumain activity and the nuclear counter-stain (right panel). Scale bar represents 10 µm. HCT116 cells incubated without substrate, or with substrate and cystatin E/M, showed no signals ( respectively). (C and D) Legumain activity (green) in cryosections from subcutaneous xenografts with nuclei stained with DAPI (red) and analyzed by confocal laser scanning microscopy. Subcutaneous xenograft from HCT116 cells (C) showed similar results as in cultured cells with intense granulated activity (gray arrow) although less distinct activity in the cytoplasm (blue arrow) and within the nucleus (yellow arrow) was also observed. However, in the subcutaneous xenograft of SW620 cells (D) majorly diffuse legumain activity was observed in the cell cytoplasm (blue arrow), while in the nucleus this was more concentrated (yellow arrow). Scale bars represent 50 µm.

Article Snippet: Control slides were prepared using buffer without substrate, the epoxy inhibitor E64 (Sigma) at a final concentration of 1 μM or human recombinant cystatin E/M (R&D Systems, 1286-PI) at a final concentration of 0.1 μM.

Techniques: In Situ, Activity Assay, Fluorescence, Microscopy, Imaging, Incubation, Recombinant, Staining, Confocal Laser Scanning Microscopy, Cell Culture

Journal: PLoS ONE

Article Title: Nuclear Legumain Activity in Colorectal Cancer

doi: 10.1371/journal.pone.0052980

Figure Lengend Snippet: (A) Immunoblots showing the cleavage of intact (lane 10) recombinant human histone H3.1 in a dose dependent manner by purified mature 36 kDa bovine legumain (bovLeg, lane 1–3) and auto-activated intermediate form (46 kDa) of recombinant human legumain (rhLeg, lane 6–8). The addition of recombinant human cystatin E/M (lane 4 and 8) efficiently blocked legumain activity and resulted in almost complete rescue of histone H3.1 from proteolytic cleavage. Uncut immunoblots . (B) Immunoblot of histone H3.1 showing the dose-dependent production of a 12 kDa cleavage product after incubation of recombinant histone H3.1 with fully mature 36 kDa bovine legumain in a buffer with pH 7.0 (lane 1–3). Addition of recombinant human cystatin E/M efficiently blocked legumain activity and resulted in virtually no formation of the 12 kDa cleavage product (lane 4).

Article Snippet: Control slides were prepared using buffer without substrate, the epoxy inhibitor E64 (Sigma) at a final concentration of 1 μM or human recombinant cystatin E/M (R&D Systems, 1286-PI) at a final concentration of 0.1 μM.

Techniques: Western Blot, Recombinant, Purification, Activity Assay, Incubation

Journal: Scientific Reports

Article Title: Staphylococcus aureus induces DNA damage in host cell

doi: 10.1038/s41598-019-44213-3

Figure Lengend Snippet: S. aureus induce 8-oxoG DNA lesion in the host cells. Either HeLa or MG-63 cells were exposed to S. aureus MW2 strain at MOI 1:50 for 2 h followed by antibiotic treatment as described. As the positive control 50 µM etoposide was used. After 6 h and 20 h of post-infection cells were immunostained with mouse anti 8-oxoG DNA lesion antibody, followed by incubation with m-IgGκ BP-FITC at dilution 1:50 for 2 h at room temperature (green staining, the red arrow). Nuclear DNA were labeled with DAPI (blue staining). Samples were viewed with Leica SP8 laser-scanning microscope equipped with immersion objective 63× plan Apo-NA 1.4 driven by the LAS software. Scale bar: 5 µm.

Article Snippet: For 8-oxoG immunostaining cells were incubated with mouse anti-8-oxo DNA lesion antibody (483.15: sc-130914, Santa Cruz, USA),(1:100), followed by incubation with mouse IgGκ light chain binding protein conjugated to fluorescein isothiocyanate (sc-516140, Santa Cruz, USA) (1:50).

Techniques: Positive Control, Infection, Incubation, Staining, Labeling, Laser-Scanning Microscopy, Software