Journal: International Journal of Molecular Sciences

Article Title: Activatable Peptides for Rapid and Simple Visualization of Protease Activity Secreted in Living Cells

doi: 10.3390/ijms23031605

Figure Lengend Snippet: Schematic of peptide-permeable cell imaging in response to MMP2 activity. After treatment with FITC-labeled peptides, an MMP2-positive (MMP2-secreted) cell shows a strong fluorescence due to intracellular delivery of cleaved peptides ( left ), whereas an MMP2-negative cell ( left ) does not generate fluorescence due to the lower cell-permeability of the peptide ( right ).

Article Snippet: MMP2/9 inhibitor ((2R)-2-[(4-biphenylylsulfonyl)amino]-3-phenylpropionic acid), gelatin from porcine skin, and Triton X-100 were purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Imaging, Activity Assay, Labeling, Fluorescence, Permeability

Journal: International Journal of Molecular Sciences

Article Title: Activatable Peptides for Rapid and Simple Visualization of Protease Activity Secreted in Living Cells

doi: 10.3390/ijms23031605

Figure Lengend Snippet: Expression and activity assays of MMP2 and MMP9 in two cell lines (HT-29 and HT-1080). ( A ) Gel electrophoresis and ( B ) real-time PCR showing mRNA expressions of proMMP2 and proMMP9 relative to that of GAPDH in HT-29 and HT-1080. The asterisk denotes significant difference between HT-29 and HT-1080 (* p < 0.05, *** p < 0.001, Student’s t test, n = 3). ( C ) Western blot and ( D ) gelatin zymographic images of (pro)MMP2 and (pro)MMP9 in the CM of HT-29 or HT-1080. Active MMP2 or MMP9 enzyme was loaded into the first lane.

Article Snippet: MMP2/9 inhibitor ((2R)-2-[(4-biphenylylsulfonyl)amino]-3-phenylpropionic acid), gelatin from porcine skin, and Triton X-100 were purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Activity Assay, Nucleic Acid Electrophoresis, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Activatable Peptides for Rapid and Simple Visualization of Protease Activity Secreted in Living Cells

doi: 10.3390/ijms23031605

Figure Lengend Snippet: Fluorescent gel image used to verify the cleavage of Pep1 and Pep2 due to MMP activity. Pep1 (lanes 1–3) or Pep2 (lanes 4–6) was loaded onto the agarose gel in the absence of enzymes (lanes 1 and 4) and in the presence of active MMP2 (lanes 2 and 5) or active MMP9 (lanes 3 and 6). The FITC-specific fluorescent band regions are indicated by arrows on the left side of the image, which corresponds to cleaved Pep1, Pep1, and Pep2 (from top to bottom). The agarose gel was exposed to LED irradiation to obtain the FITC-emitted image.

Article Snippet: MMP2/9 inhibitor ((2R)-2-[(4-biphenylylsulfonyl)amino]-3-phenylpropionic acid), gelatin from porcine skin, and Triton X-100 were purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Activity Assay, Agarose Gel Electrophoresis, Irradiation

Journal: International Journal of Molecular Sciences

Article Title: Activatable Peptides for Rapid and Simple Visualization of Protease Activity Secreted in Living Cells

doi: 10.3390/ijms23031605

Figure Lengend Snippet: ( A ) Overlay (bright-field and fluorescence) and ( B ) fluorescence confocal images of HT-1080 (MMP2-positive cells) and HT-29 (MMP2-negative cells) treated with different peptides (Pep1, Pep2, or Pep3). A 10 μM peptide was added to the serum-free culture media of the cells. Images were collected from 2 h after treatment. Scale bar: 50 μm.

Article Snippet: MMP2/9 inhibitor ((2R)-2-[(4-biphenylylsulfonyl)amino]-3-phenylpropionic acid), gelatin from porcine skin, and Triton X-100 were purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Fluorescence

Journal: Journal of Neuroinflammation

Article Title: Rottlerin, a natural polyphenol compound, inhibits upregulation of matrix metalloproteinase-9 and brain astrocytic migration by reducing PKC-δ-dependent ROS signal

doi: 10.1186/s12974-020-01859-5

Figure Lengend Snippet: Phorbol 12-myristate 13-acetate (PMA), a PKC activator, induces MMP-9 expression, but not MMP-2, in brain astrocytes (RBA). a Time dependence of PMA increase of MMP-9 expression. RBA cells were treated with 1 μM PMA for the indicated time intervals. b Concentration dependence of PMA-induced MMP-9 expression. Cells were treated with various concentrations of PMA (0.01, 0.1, 1, and 10 μM) for 24 h. c Time dependence of PMA-induced MMP 9 mRNA expression. Cells were treated with 1 μM PMA for the indicated times. The conditioned media, cell lysates, and total RNA were collected and analyzed by gelatin zymography (MMP2/9), Western blot (GAPDH, as an internal control), and RT-PCR (MMP-9 and β-actin) as described under the “Methods” section. The intensity of zymographic ( a , b ) and PCR product ( c ) bands was quantitated by scanning densitometry and expressed as fold of untreated control. Data are expressed as the mean ± SEM ( N = 3). * P < 0.05; ** P < 0.01, as compared with the respective values of untreated control. The image represents one of three individual experiments

Article Snippet: Rottlerin, apocynin, PD98059, tanshinone IIA (TSIIA), and MMP2/9 inhibitor (2/9i) were from Biomol (Plymouth Meeting, PA).

Techniques: Expressing, Concentration Assay, Zymography, Western Blot, Reverse Transcription Polymerase Chain Reaction