Journal: Human Reproduction (Oxford, England)

Article Title: Chronic hyperandrogenemia in the presence and absence of a western-style diet impairs ovarian and uterine structure/function in young adult rhesus monkeys

doi: 10.1093/humrep/dex338

Figure Lengend Snippet: Photomicrographs illustrating immunohistochemical staining for estrogen receptor 1 (ESR1), progesterone receptor (PGR) MMP26, TIMP3, Ki-67 and androgen receptor (AR) in the endometrial functionalis zone of the macaque uterus from representative females in each treatment group (C, T, WSD, T+WSD). Brown staining denotes positive expression of proteins. Sections are counterstained with hematoxylin (blue) staining. ESR1, PGR, Ki-67 and AR staining is nuclear, while MMP26 and TIMP3 show cytoplasmic localization. Inset shows a negative control with an irrelevant antibody (Anti-Br(d)U). TIMP3 staining was localized to the predecidual cells around the spiral arteries.

Article Snippet: Antibodies used were against estrogen receptor 1 (ESR1,ER-ID5; Cat#: MS-354-P, Thermo Fisher Scientific), progesterone receptor (PGR, Cat#: Ms-298-P, 1 μg, Lab Vision/NeoMarkers, Fremont, CA, USA), androgen receptor (AR-F39 (Cat#: MU256, 1/50, BioGenex, Fremont, CA, USA), Ki67 (Cat#: MU370-UC, 1/200, BioGenex), MMP26 (Cat# ab57636; Abcam, Cambridge, MA, USA) and TIMP3 (Cat#:MAB973, R&D Systems, Inc. Minneapolis, MN, USA).

Techniques: Immunohistochemical staining, Staining, Expressing, Negative Control

Journal: Human Reproduction (Oxford, England)

Article Title: Chronic hyperandrogenemia in the presence and absence of a western-style diet impairs ovarian and uterine structure/function in young adult rhesus monkeys

doi: 10.1093/humrep/dex338

Figure Lengend Snippet: Expression of ESR1, PGR, MMP26 and TIMP3 mRNAs as detected by qRT-PCR in endometrial biopsies collected from macaques in the mid-luteal phase of the cycle. Significant (P < 0.05) differences between individual treatment groups are denoted by different uppercase letters above columns.

Article Snippet: Antibodies used were against estrogen receptor 1 (ESR1,ER-ID5; Cat#: MS-354-P, Thermo Fisher Scientific), progesterone receptor (PGR, Cat#: Ms-298-P, 1 μg, Lab Vision/NeoMarkers, Fremont, CA, USA), androgen receptor (AR-F39 (Cat#: MU256, 1/50, BioGenex, Fremont, CA, USA), Ki67 (Cat#: MU370-UC, 1/200, BioGenex), MMP26 (Cat# ab57636; Abcam, Cambridge, MA, USA) and TIMP3 (Cat#:MAB973, R&D Systems, Inc. Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Long noncoding RNA DANCR promotes invasion of prostate cancer through epigenetically silencing expression of TIMP2/3

doi: 10.18632/oncotarget.9350

Figure Lengend Snippet: ( A ) Effect of DANCR knockdown on the expression of thirty-seven invasion associated genes in C4-2B cells, as detected by RT-qPCR assay. ( B , C ) Knockdown of DANCR increases the expression of TIMP2/3 mRNA and protein in C4-2B and CW22RV1 cells, as detected by RT-qPCR assay and western blot analysis. ( D ) Representation of the TImP2/3 promoter region as mapped by PCR analysis and ChIP assay. The bent arrow represents the transcription start sites (+1). The lines below the TIMP2/3 locus represent the regions amplified by PCR. ( E ) Knockdown of DANCR decreased the binding of EZH2 on the promoter of TIMP2/3. Immuno-precipitated DNA was analyzed by PCR with specific primer sets. Chromatin obtained from C4-2B and CW22RV1 cells were immune-precipitated using antibodies to EZH2, histone H3 (H3) and IgG. Each ChIP experiment was repeated at least three times and a representative experiment is shown. ( F ) Knockdown of DANCR decreased the tri-methyl-histone H3K27 on the promoter of TIMP2/3. Immuno-precipitated DNA was analyzed by PCR with specific primer sets. Chromatin obtained from C4-2B and CW22RV1cells were immune-precipitated using antibodies to tri-methyl-histone H3K27 (3meH3K27), histone H3 (H3) and IgG. Each experiment was repeated at least three times and a representative experiment is shown. ( G ) Knockdown of DANCR decreased the binding of EZH2 on the promoter of TIMP2/3, as detected by oligonucleotides pull down assays. P1: TIMP2-set2; P2: TIMP2-set3; P3: TIMP3-set1; P4: TIMP3-set2; P5: TIMP3-set3. ( H ) DANCR was pulled down by the oligonucleotides with the same sequence of the promoters of TIMP2/3 (P1-P5). Control: The DNA solution TE buffer was used as negative control.

Article Snippet: Then membranes were blocked with 5% skim milk at room temperature for 1 hour and then incubated with primary antibodies against GAPDH (Kangchen, Shanghai, China), TIMP2, TIMP3 and androgen receptor (AR) (Santa Cruz, Dallas, TX, USA) at 4°C overnight, followed by TBST wash and 1 hour incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Amplification, Binding Assay, Sequencing, Control, Negative Control

Journal: Molecular Systems Biology

Article Title: Global remodelling of cellular microenvironment due to loss of collagen VII

doi: 10.1038/msb.2013.17

Figure Lengend Snippet: Decrease in protease activity in C7-deficient fibroblasts. ( A ) Western blot of MMP2 and MMP14 of ECM. ( B ) The blue rounds represent the intact collagen I matrix. Upon degradation of the matrix, the staining becomes white. In contrast to the RDEB cells (pt), control cells (ctrl) degrade the matrix, and the degradation is stimulated by the addition of TNFα. Inhibitor profiling demonstrates that the degradation is mediated by metalloproteases, since TAPIO, D-RVKR-CMK and TIMP3, but not serine and cystein protease inhibitors leupeptin, SBTI, and PSMF prevent it. ConA, inhibiting lysosomal degradation, leads only in control cells to enhanced degradation. ( C ) ConA leads to enhanced MMP2 activity in control cells as analysed by zymography. Western blot analysis reveals impaired MMP14 turnover in patient cells compared with control cells. Numbers indicating fold change as compared with untreated control. Samples were normalised to protein amount. ( D ) ECM proteins of RDEB (pt1, pt3) and control (ctrl2, ctrl3) cells were separated by SDS–PAGE and analysed by MS. The total number of detected MMP14 peptides in one sample was set to 100% and the distribution of peptides was mapped to respective gel slices. MMP14 appears to be shed in RDEB cells and not in control cells, as only ∼50% of the MMP14 peptides were detected in the 60-kDa gel slice harbouring full-length MMP14 in RDEB cells in contrast to almost 100% in control cells. ( E ) Schematic representation of the MMP14 molecule. Peptides derived from control cells (green) and RDEB cells (yellow) were mapped to the MMP14 molecule. Combined results of two biological replicates are shown. ( F ) Model of altered MMP14 turnover. Under healthy control conditions, C7 is present and active in the extracellular environment. MMP14 recycling and degradation is ensured to maintain degradation of collagen I and activation of MMP2. In RDEB, the ECM lacks C7, leading to an accumulation of MMP14, MMP2 and TIMP3. MMP14 and MMP2 activity is impaired due to the presence of inhibitors like TIMP3 and increased shedding of MMP14. Additionally, MMP14 recycling is perturbed further amplifying its accumulation.

Article Snippet: Following concentrations were used: 10 ng/ml TNF-α (Peprotech, Hamburg, Germany), 2 nM ConA (Peprotech), 25 μM TAPI-O (CalBiochem), 1 mM PMSF (Sigma-Aldrich), 1 μM SBTI (Sigma-Aldrich), 1 μg/ml Leupeptin (Sigma-Aldrich), 50 μM Furin Inhibitor I (Decanoyl-Arg-Val-Lys-Arg-CMK, Calbiochem/Merck, Darmstadt, Germany), 100 nM human recombinant TIMP3 (R&D Systems).

Techniques: Activity Assay, Western Blot, Staining, Control, Zymography, SDS Page, Derivative Assay, Activation Assay