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:

Article Title: Active MMP-2 Activity Discriminates Colonic Mucosa, Adenomas with and without High Grade Dysplasia and Cancers

doi: 10.1016/j.humpath.2010.08.021

Figure Lengend Snippet: Normal, adenoma, high grade dysplasia and cancer: Mean MMP activity/mg protein expressed in 3 types of units for patient-matched tissues from 30 individuals (no FAP cases)

Article Snippet: In addition, to confirm identity of specific gel bands and to standardize activity measurements, purified active gelatinase controls for active MMP-2 (human recombinant active MMP-2), pro-MMP-2 (human synovial fibroblast proenzyme MMP-2) which is separated from a non-covalent 1:1 complex with TIMP-2 and activated in the presence of SDS during gel electrophoresis ( 14 ) and human neutrophil MMP-9 (all from Calbiochem, La Jolla, CA USA) were electrophoresed next to the tissue extracts, as detailed below.

Techniques: Activity Assay

Journal:

Article Title: Active MMP-2 Activity Discriminates Colonic Mucosa, Adenomas with and without High Grade Dysplasia and Cancers

doi: 10.1016/j.humpath.2010.08.021

Figure Lengend Snippet: 1a. MMP-2 and MMP-9 activities in 8 matched pairs of normal and cancer tissues. Gelatin zymograms for eight pairs of patient-matched normal mucosa (N) and colorectal cancer (C) extracts with Coomassie blue stained protein markers (M) of defined molecular mass to the left. Active MMP-2 was detected as a 60 kD band showing stronger activity in all cancer samples compared to matched normal mucosa. Pro-MMP-2, detected as a 65 kD band, was present in all samples, with higher activity in 6/8 cancers compared to matched normal mucosa. Active MMP-9 activity was detected as a weak 80 kD band in two normal mucosa samples (N29 and N130). Pro-MMP-9 activity was detected as a 97 kD band demonstrating a wide range of variation in activity levels from almost non-detectable (N129) to very high levels (C331) and increased activity in 5/8 cancer samples compared to normal mucosa. Cancer stage is designated above each cancer tissue (Stage I, S I; Stage II, S II; Stage III, S III; Stage IV, S IV).

Article Snippet: In addition, to confirm identity of specific gel bands and to standardize activity measurements, purified active gelatinase controls for active MMP-2 (human recombinant active MMP-2), pro-MMP-2 (human synovial fibroblast proenzyme MMP-2) which is separated from a non-covalent 1:1 complex with TIMP-2 and activated in the presence of SDS during gel electrophoresis ( 14 ) and human neutrophil MMP-9 (all from Calbiochem, La Jolla, CA USA) were electrophoresed next to the tissue extracts, as detailed below.

Techniques: Staining, Activity Assay

Journal:

Article Title: Active MMP-2 Activity Discriminates Colonic Mucosa, Adenomas with and without High Grade Dysplasia and Cancers

doi: 10.1016/j.humpath.2010.08.021

Figure Lengend Snippet: MMP-2 and -9 activities for each of four different types of colorectal tissues in patient-matched sets of tissue extracts (Normal colon mucosa, n = 30; Adenomas without HGD (designated Adenoma), n = 18; Adenomas with HGD (designated HGD), n = 16; Cancer, n = 11) are graphed in serial dilution units/mg protein. Median activity levels are represented by a solid vertical line intersecting the central box of each plot.

Article Snippet: In addition, to confirm identity of specific gel bands and to standardize activity measurements, purified active gelatinase controls for active MMP-2 (human recombinant active MMP-2), pro-MMP-2 (human synovial fibroblast proenzyme MMP-2) which is separated from a non-covalent 1:1 complex with TIMP-2 and activated in the presence of SDS during gel electrophoresis ( 14 ) and human neutrophil MMP-9 (all from Calbiochem, La Jolla, CA USA) were electrophoresed next to the tissue extracts, as detailed below.

Techniques: Serial Dilution, Activity Assay

Journal:

Article Title: Active MMP-2 Activity Discriminates Colonic Mucosa, Adenomas with and without High Grade Dysplasia and Cancers

doi: 10.1016/j.humpath.2010.08.021

Figure Lengend Snippet: Parameter estimates (robust standard errors) and approximate p -values from two fitted multinomial logit models that discriminate among colorectal tissue types using active MMP-2, pro-MMP-2 and pro-MMP-9 activity levels

Article Snippet: In addition, to confirm identity of specific gel bands and to standardize activity measurements, purified active gelatinase controls for active MMP-2 (human recombinant active MMP-2), pro-MMP-2 (human synovial fibroblast proenzyme MMP-2) which is separated from a non-covalent 1:1 complex with TIMP-2 and activated in the presence of SDS during gel electrophoresis ( 14 ) and human neutrophil MMP-9 (all from Calbiochem, La Jolla, CA USA) were electrophoresed next to the tissue extracts, as detailed below.

Techniques: Activity Assay

Journal:

Article Title: Active MMP-2 Activity Discriminates Colonic Mucosa, Adenomas with and without High Grade Dysplasia and Cancers

doi: 10.1016/j.humpath.2010.08.021

Figure Lengend Snippet: Estimated probabilities of declaring a colorectal tissue sample as either normal, adenoma without HGD, adenoma with HGD or cancer graphed with respect to levels of active MMP-2 activity (SDU/mg) in that sample. At any given value a comparison of these probabilities permits prediction of the most probable diagnosis of the tissue as one of these four tissue types.

Article Snippet: In addition, to confirm identity of specific gel bands and to standardize activity measurements, purified active gelatinase controls for active MMP-2 (human recombinant active MMP-2), pro-MMP-2 (human synovial fibroblast proenzyme MMP-2) which is separated from a non-covalent 1:1 complex with TIMP-2 and activated in the presence of SDS during gel electrophoresis ( 14 ) and human neutrophil MMP-9 (all from Calbiochem, La Jolla, CA USA) were electrophoresed next to the tissue extracts, as detailed below.

Techniques: Activity Assay