Journal: PLoS ONE

Article Title: Matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases 1 (TIMP-1) are localized in the nucleus of retinal Müller glial cells and modulated by cytokines and oxidative stress

doi: 10.1371/journal.pone.0253915

Figure Lengend Snippet: (A) MIO-M1 cells were treated or untreated with IL-1β and TNF-α, at 10 ng/mL, alone or in combination, for 24 h, and then subjected to MTT assay to measure viable cell densities. Control refers to untreated samples. Relative cell densities are presented as % mean ± SE (n = 3), with Control set as 100%. **, p<0.01 (vs. Control) (B) The cells were treated in the same way as in (A), and then CMs were collected, to perform gelatin zymography assay. (Top) CMs of the same volume from each treatment was subjected to gelatin zymography. One representative zymogram is shown to visualize secreted MMPs. Recombinant human pro-MMP-2 and pro-MMP-9 (Pro-MMP-2+Pro-MMP-9) were applied to the gel before electrophoresis as a control. (Bottom) The density of each MMP band was quantified using Image J software. The individual values of MMP band densities were normalized by the corresponding MTT absorbance values (n = 3). ns, not significant (p>0.05 vs. Control); *, p<0.05 (vs. Control) (C) Secreted TIMP-1 levels in the CMs were measured using TIMP-1 ELISA assay (n = 3), and then normalized by the corresponding MTT absorbance values. *, p<0.05 (IL-1β vs. TNF-α).

Article Snippet: Human interleukin (IL)-1β, tumor necrosis factor (TNF)-α, pro-MMP-9 and pro-MMP-2 were purchased from R&D Systems (Minneapolis, MN).

Techniques: MTT Assay, Zymography Assay, Zymography, Recombinant, Electrophoresis, Software, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases 1 (TIMP-1) are localized in the nucleus of retinal Müller glial cells and modulated by cytokines and oxidative stress

doi: 10.1371/journal.pone.0253915

Figure Lengend Snippet: (A) MIO-M1 cells were cultured in the presence of H 2 O 2 at indicated concentrations in the regular (10% FBS) or FBS-free media, for 24 h, and then subjected to MTT assay to measure viable cell densities. Relative cell densities are presented as % mean ± SE (n = 3), with control (0μM) set as 100%. (B) The cells were treated in the same way as in (A), and then CMs were collected, to perform gelatin zymography and ELISA assay. (Top) the CMs of the same volume from each treatment was subjected to gelatin zymography. One representative zymogram is shown to visualize secreted MMPs. A mixture of recombinant human pro-MMP-2 and pro-MMP-9 (Pro-MMP-2+Pro-MMP-9) were applied to the gel prior to electrophoresis as a MMPs marker control. (Bottom) The density of each MMP band was quantified using Image J software (n = 3). The individual values of MMP band densities were normalized by the corresponding MTT absorbance values. (C) Secreted TIMP-1 levels in the same CMs were measured using TIMP-1 ELISA assay (n = 3). The individual ELISA values were normalized by the corresponding MTT absorbance values. ns, not significant (p>0.05, vs. 0μM). (D) MIO-M1 cells were cultured in the presence of 300 μM H 2 O 2 for 24 h, and then subjected to IHC confocal microscopy, for dual IHC staining with MMP-9 (red) and TIMP-1 (red) antibodies. Insets in TIMP-1+DAPI micrograms are enlarged, and arrows point to the bigger nuclei with increased TIMP-1 speckles. (E) Nuclear sizes in the micrograms of the cells treated with increasing concentrations of H 2 O 2 were measured, using Image J software. Relative nuclear sizes are presented as % mean ± SE. ns, not significant (p>0.05 vs. 0μM); ***, p<0.001 (vs. vs. 0μM; ****, p<0.0001 (vs. 0μM); #, p<0.001 (vs. 100μM); ‡, p<0.01 (vs. 300μM). The numbers of randomly selected nuclei measured were 43 (0μM), 55 (100μM), 36 (300μM), and 37 (600μM). (F) β-actin (red) antibody was used to perform a confocal microscopy after 300μM H 2 O 2 treatment for 24h. Representative micrographs of untreated (0μM) and treated (300μM) groups are presented. bars, 50 μm.

Article Snippet: Human interleukin (IL)-1β, tumor necrosis factor (TNF)-α, pro-MMP-9 and pro-MMP-2 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, MTT Assay, Zymography, Enzyme-linked Immunosorbent Assay, Recombinant, Electrophoresis, Marker, Software, Confocal Microscopy, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Cell-Penetrating Doxorubicin Released from Elastin-Like Polypeptide Kills Doxorubicin-Resistant Cancer Cells in In Vitro Study

doi: 10.3390/ijms22031126

Figure Lengend Snippet: MMP-2 digestion of ELP-mmpL-CPP. ( A ) ELP-mmpL-CPP-rhodamine and ELP-CPP-rhodamine were incubated with MMP-2 for 4 h in ZnCl 2 buffer (pH 7). ( B ) MMP-2 incubation of the constructs produced ELP-CPP-Dox (63 kDa, upper band) and CPP-rhodamine (3 kDa, lower band). Left panel: silver-stained gel; Right panel: fluorescence-scanned gel. Dox: 6-maleimidocaproyl amide derivative of doxorubicin; mmpL: MMP substrate.

Article Snippet: Recombinant human pro-MMP-2 (Enzo life science) was activated with 2.5 mM 4-aminophenylmercuric acetate at 37 °C for 2 h. Then, 1 µg of each ELP construct was incubated with the pre-activated MMP-2 (10 pmol) for 4 h in a reaction buffer (50 mM Tris, 200 mM NaCl, 10 mM CaCl 2 , and 10 mM ZnCl 2 , pH 7.5).

Techniques: Incubation, Construct, Produced, Staining, Fluorescence