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: Materials Today Bio

Article Title: Cationic microbubble loading hSIRT3 and hTIMP3 optimize cardiac-targeted delivery and myocardial protection in the porcine MI/R model

doi: 10.1016/j.mtbio.2025.102234

Figure Lengend Snippet: Characteristics of myocardial and cardiac function in a porcine MI/R injury model. a. Dynamic ECG in the establishment of porcine MI/R injury model. b. the level of LVEF, LVFS at baseline and days 3, 7, 14 after MI/R injury. c. Representative images for CD11b and Col1a1 expressions in IZ myocardium of the Sham group and on days 3, 7, and 14 after MI/R injury (scale bar = 100 μm). d. The protein expressions and quantitative analysis of endogenous SIRT3 and TIMP3 in IZ, BZ, and RZ in the Sham group and on days 3, 7, and 14 after MI/R injury (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs. Sham group). Data are presented as the Means ± SD (n = 3). ECG: Electrocardiogram. LV-EF: Left ventricular ejection fraction. LV-FS: Left ventricular fractional shortening. IZ: Infarction zone. BZ: Border zone. RZ: Remote zone.

Article Snippet: The primary antibodies against hSIRT3 (1:1,000, ab217319, Abcam), hTIMP3 (1:1,000, ab276134, Abcam), SIRT3 (1:1,000, 10099-1-AP, Proteintech), TIMP3 (1:1,000, 10858-1-AP, Proteintech), MnSOD (1:5,000, 24127-1-AP, Proteintech), CAT (1:2,000, 21260-1-AP, Proteintech), MMP2 (1:500, 10373-2-AP, Proteintech), MMP9 (1:1,000, 10375-2-AP, Proteintech), caspase-3 (1:1,000, 66470-2-Ig, Proteintech), Bcl2 (1:1,000, ab32124, Abcam) and Bax (1:5,000, 50599-2-Ig, Proteintech) were incubated with the corresponding PVDF membranes overnight at 4 °C.

Techniques:

Journal: Materials Today Bio

Article Title: Cationic microbubble loading hSIRT3 and hTIMP3 optimize cardiac-targeted delivery and myocardial protection in the porcine MI/R model

doi: 10.1016/j.mtbio.2025.102234

Figure Lengend Snippet: Characteristics of CMBs. a. Schematic diagram of CMB preparation. b. The appearance of CMBs. c. Microscopic morphology of CMBs detected by TEM (scale bar = 100 nm). d. The distributions of diameter and (e) surface charge of CMBs. f. The morphology of CMBs/vector labeled with PI stain (red). Scale bar = 50 μm. g. The encapsulation efficiency of CMBs for different doses of hSIRT3 or hTIMP3 genes. h. The loading rate of CMBs with hSIRT3 and hTIMP3 plasmids detected by flow cytometry. Data are presented as the Means ± SD (n = 3). CMBs: Cationic microbubbles. TEM: Transmission electron microscopy. hSIRT3: human SIRT3. hTIMP3: human TIMP3. ∗∗∗ P < 0.001. n.s.: not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies against hSIRT3 (1:1,000, ab217319, Abcam), hTIMP3 (1:1,000, ab276134, Abcam), SIRT3 (1:1,000, 10099-1-AP, Proteintech), TIMP3 (1:1,000, 10858-1-AP, Proteintech), MnSOD (1:5,000, 24127-1-AP, Proteintech), CAT (1:2,000, 21260-1-AP, Proteintech), MMP2 (1:500, 10373-2-AP, Proteintech), MMP9 (1:1,000, 10375-2-AP, Proteintech), caspase-3 (1:1,000, 66470-2-Ig, Proteintech), Bcl2 (1:1,000, ab32124, Abcam) and Bax (1:5,000, 50599-2-Ig, Proteintech) were incubated with the corresponding PVDF membranes overnight at 4 °C.

Techniques: Plasmid Preparation, Labeling, Staining, Encapsulation, Flow Cytometry, Transmission Assay, Electron Microscopy

Journal: Materials Today Bio

Article Title: Cationic microbubble loading hSIRT3 and hTIMP3 optimize cardiac-targeted delivery and myocardial protection in the porcine MI/R model

doi: 10.1016/j.mtbio.2025.102234

Figure Lengend Snippet: Cardiac-targeted delivery of hSIRT3 and hITMP3 plasmids by UTMD. a. Schematic diagram of UTMD procedure for cardiac-targeted delivery of hSIRT3 and hITMP3 plasmids. b. Representative ultrasound contrast images of imaging and destructing CMBs/hSIRT3 or CMBs/hTIMP3 in the heart. c. Representative images of GFP expression in the myocardium and quantitative analysis of GPF expression among the groups. d. Protein expressions and quantitative analysis of hSIRT3 and hTIMP3 among the groups. e. Protein expressions and quantitative analysis of hSIRT3 and hTIMP3 among RZ, BZ and IZ myocardium. Data are presented as the Means ± SD (n = 3). UTMD: Ultrasound-targeted microbubble destruction. CMBs: Cationic microbubbles. hSIRT3: human SIRT3. hTIMP3: human TIMP3. IZ: Infarction zone. BZ: Border zone. RZ: Remote zone. ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The primary antibodies against hSIRT3 (1:1,000, ab217319, Abcam), hTIMP3 (1:1,000, ab276134, Abcam), SIRT3 (1:1,000, 10099-1-AP, Proteintech), TIMP3 (1:1,000, 10858-1-AP, Proteintech), MnSOD (1:5,000, 24127-1-AP, Proteintech), CAT (1:2,000, 21260-1-AP, Proteintech), MMP2 (1:500, 10373-2-AP, Proteintech), MMP9 (1:1,000, 10375-2-AP, Proteintech), caspase-3 (1:1,000, 66470-2-Ig, Proteintech), Bcl2 (1:1,000, ab32124, Abcam) and Bax (1:5,000, 50599-2-Ig, Proteintech) were incubated with the corresponding PVDF membranes overnight at 4 °C.

Techniques: Imaging, Expressing

Journal: Materials Today Bio

Article Title: Cationic microbubble loading hSIRT3 and hTIMP3 optimize cardiac-targeted delivery and myocardial protection in the porcine MI/R model

doi: 10.1016/j.mtbio.2025.102234

Figure Lengend Snippet: Exogenous hSIRT3 and hTIMP3 genes exerted their downstream signaling regulation and inhibited the infiltration of inflammatory cells. a. Flowchart of short-term effects of exogenous hSIRT3 and hTIMP3 genes on myocardial injury. b. Protein expressions and quantitative analysis of CAT and MnSOD among the different groups. c. Protein expressions and quantitative analysis of MMP2 and MMP9 among the different groups. d. Representative images of CD11b expression in the myocardium and quantitative analysis of CD11b expression among the different groups (scale bar = 100 μm). Data are presented as the Means ± SD (n = 3). hSIRT3: human SIRT3. hTIMP3: human TIMP3. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. n.s.: not significant.

Article Snippet: The primary antibodies against hSIRT3 (1:1,000, ab217319, Abcam), hTIMP3 (1:1,000, ab276134, Abcam), SIRT3 (1:1,000, 10099-1-AP, Proteintech), TIMP3 (1:1,000, 10858-1-AP, Proteintech), MnSOD (1:5,000, 24127-1-AP, Proteintech), CAT (1:2,000, 21260-1-AP, Proteintech), MMP2 (1:500, 10373-2-AP, Proteintech), MMP9 (1:1,000, 10375-2-AP, Proteintech), caspase-3 (1:1,000, 66470-2-Ig, Proteintech), Bcl2 (1:1,000, ab32124, Abcam) and Bax (1:5,000, 50599-2-Ig, Proteintech) were incubated with the corresponding PVDF membranes overnight at 4 °C.

Techniques: Expressing

Journal: Materials Today Bio

Article Title: Cationic microbubble loading hSIRT3 and hTIMP3 optimize cardiac-targeted delivery and myocardial protection in the porcine MI/R model

doi: 10.1016/j.mtbio.2025.102234

Figure Lengend Snippet: Exogenous hSIRT3 and hTIMP3 genes inhibited apoptosis of myocardium. a. Representative images of IL-1β, IL-6 and TNF-α expressions in IZ myocardium and quantitative analysis (b) among the different groups (scale bar = 100 μm). c. Protein expressions and quantitative analysis of caspase-3, Bcl2, and Bax among the different groups. d. Representative images of TUNEL-positive cells in the myocardium and quantitative analysis of TUNEL-positive cells among the different groups (scale bar = 100 μm). Data are presented as the Means ± SD (n = 3). hSIRT3: human SIRT3. hTIMP3: human TIMP3. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. n.s.: not significant.

Article Snippet: The primary antibodies against hSIRT3 (1:1,000, ab217319, Abcam), hTIMP3 (1:1,000, ab276134, Abcam), SIRT3 (1:1,000, 10099-1-AP, Proteintech), TIMP3 (1:1,000, 10858-1-AP, Proteintech), MnSOD (1:5,000, 24127-1-AP, Proteintech), CAT (1:2,000, 21260-1-AP, Proteintech), MMP2 (1:500, 10373-2-AP, Proteintech), MMP9 (1:1,000, 10375-2-AP, Proteintech), caspase-3 (1:1,000, 66470-2-Ig, Proteintech), Bcl2 (1:1,000, ab32124, Abcam) and Bax (1:5,000, 50599-2-Ig, Proteintech) were incubated with the corresponding PVDF membranes overnight at 4 °C.

Techniques: TUNEL Assay

Journal: Materials Today Bio

Article Title: Cationic microbubble loading hSIRT3 and hTIMP3 optimize cardiac-targeted delivery and myocardial protection in the porcine MI/R model

doi: 10.1016/j.mtbio.2025.102234

Figure Lengend Snippet: Exogenous hSIRT3 and hTIMP3 genes promoted revascularization after myocardial injury. a. Flowchart of long-term effects of exogenous hSIRT3 and hTIMP3 genes on myocardial injury. b. Representative images of α-SMA labeled arteries and (c) CD31 labeled veins in the myocardium and quantitative analysis among the different groups (scale bar = 100 μm). Data are presented as the Means ± SD (n = 3). hSIRT3: human SIRT3. hTIMP3: human TIMP3. ∗∗ P < 0.01, ∗∗∗ P < 0.001. n.s.: not significant.

Article Snippet: The primary antibodies against hSIRT3 (1:1,000, ab217319, Abcam), hTIMP3 (1:1,000, ab276134, Abcam), SIRT3 (1:1,000, 10099-1-AP, Proteintech), TIMP3 (1:1,000, 10858-1-AP, Proteintech), MnSOD (1:5,000, 24127-1-AP, Proteintech), CAT (1:2,000, 21260-1-AP, Proteintech), MMP2 (1:500, 10373-2-AP, Proteintech), MMP9 (1:1,000, 10375-2-AP, Proteintech), caspase-3 (1:1,000, 66470-2-Ig, Proteintech), Bcl2 (1:1,000, ab32124, Abcam) and Bax (1:5,000, 50599-2-Ig, Proteintech) were incubated with the corresponding PVDF membranes overnight at 4 °C.

Techniques: Labeling

Journal: Materials Today Bio

Article Title: Cationic microbubble loading hSIRT3 and hTIMP3 optimize cardiac-targeted delivery and myocardial protection in the porcine MI/R model

doi: 10.1016/j.mtbio.2025.102234

Figure Lengend Snippet: Exogenous hSIRT3 and hTIMP3 genes suppressed myocardial fibrosis after myocardial injury. a. Representative images of collagen deposition and myocardial fibrosis of IZ stained by Masson's and SR assays and quantitative analysis among the different groups (scale bar = 100 μm). b. Dynamic values of LV-EF, LV-FS at baseline and different time points after gene therapy among the groups. c. Representative images and quantitative analysis of fibrotic scar among the different groups. Data are presented as the Means ± SD (n = 3). hSIRT3: human SIRT3. hTIMP3: human TIMP3. IZ: Infarction zone. BZ: Border zone. RZ: Remote zone. LV-EF: Left ventricular ejection fraction. LV-FS: Left ventricular fractional shortening. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. n.s.: not significant.

Article Snippet: The primary antibodies against hSIRT3 (1:1,000, ab217319, Abcam), hTIMP3 (1:1,000, ab276134, Abcam), SIRT3 (1:1,000, 10099-1-AP, Proteintech), TIMP3 (1:1,000, 10858-1-AP, Proteintech), MnSOD (1:5,000, 24127-1-AP, Proteintech), CAT (1:2,000, 21260-1-AP, Proteintech), MMP2 (1:500, 10373-2-AP, Proteintech), MMP9 (1:1,000, 10375-2-AP, Proteintech), caspase-3 (1:1,000, 66470-2-Ig, Proteintech), Bcl2 (1:1,000, ab32124, Abcam) and Bax (1:5,000, 50599-2-Ig, Proteintech) were incubated with the corresponding PVDF membranes overnight at 4 °C.

Techniques: Staining

Journal: Cancer gene therapy

Article Title: Tissue inhibitor of metalloproteinases-3 transfer suppresses malignant behaviors of colorectal cancer cells.

doi: 10.1038/cgt.2012.70

Figure Lengend Snippet: Figure 1. TIMP3 was downregulated in colon cancer tissues. (a) TIMP3 expression at mRNA level was detected in colon cancers and paired normal mucosal tissues. TIMP3 was expressed in normal mucosal tissues but not cancer tissues. (N, normal mucosal tissue and C, colon cancer tissue); (b) Western blot showed lower expression of TIMP3 in colon cancer tissues than in normal mucosa; (c) A pair of the stained tissues from the same patient to show TIMP3 expression (left: normal mucosal tissue, right: colon cancer tissue). TIMP3 expression was obvious in cytoplasm of normal mucosal cell, but was shut off in cancer tissue. (d) The quantitative analysis of the TIMP3 staining in tissue array. TIMP3 expression was much lower in cancer tissues than that in normal control tissues (compared with normal mucosa, *Po0.01).

Article Snippet: The TIMP3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Staining, Control

Journal: Cancer gene therapy

Article Title: Tissue inhibitor of metalloproteinases-3 transfer suppresses malignant behaviors of colorectal cancer cells.

doi: 10.1038/cgt.2012.70

Figure Lengend Snippet: Figure 2. Ad-TIMP3 induced cell apoptosis and suppressed cell growth. (a) TIMP3 mRNA expression was gradually upregulated with increased MOI after Ad-TIMP3 infection, whereas TIMP3 was not detectable by RT-PCR in control CT26 cells. The fold change of TIMP3 mRNA expression relative to that of 50 MOI infection was calculated. (b) By Ad-TIMP3 infection, TIMP3 protein was increased at a dose-dependent manner. To show the exogenous TIMP3-induced apoptosis, we detected the PARP cleavage. It showed the PARP cleavage occurred with the intensity of Ad-TIMP3 infection. (c) CT26 cells were transfected by Ad-TIMP3 or Ad-Null at the indicated MOI for 72 h and then subjected to annexin V-FITC assay. Percentage of apoptosis was defined by % of cells that were FITC þ/PI and FITC þ/PI þ. The experiment was performed twice independently; (d) CT26 cells were infected with Ad-TIMP3 or Ad-Null at the indicated MOI for 72 h. Trypan blue exclusion was used to determine cell number (compared with Ad-Null, *Po0.05). (e) Ad-TIMP3 infection suppressed the activity of MMP2 and MMP9 in the culture supernatant. In the gelatin zymography assay, MMP2 and MMP9 activity was suppressed in a dose-dependent manner by Ad-TIMP3.

Article Snippet: The TIMP3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Control, Transfection, Activity Assay, Zymography Assay

Journal: Cancer gene therapy

Article Title: Tissue inhibitor of metalloproteinases-3 transfer suppresses malignant behaviors of colorectal cancer cells.

doi: 10.1038/cgt.2012.70

Figure Lengend Snippet: Figure 3. Ad-TIMP3 impaired metastasis ability of cancer cells. (a) Adhesion ability of cancer cells was impaired by Ad-TIMP3 infection in a dose-dependent manner. The control virus Ad-Null did not significantly affect the adhesion ability at 50 MOI of infection. Similarly, Ad-TIMP3 decreased the migration (b) and invasion (c) ability of CT26 cells (compared with Ad-Null, *Po0.05). Representative micrographs of the transwell migration (d) and invasion (e) were shown ( 200 magnification).

Article Snippet: The TIMP3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Infection, Control, Virus, Migration

Journal: Cancer gene therapy

Article Title: Tissue inhibitor of metalloproteinases-3 transfer suppresses malignant behaviors of colorectal cancer cells.

doi: 10.1038/cgt.2012.70

Figure Lengend Snippet: Figure 4. Ad-TIMP3 infection decreased the tumor-formation ability of cancer cells. In the in invo tumor-formation assay, Ad-TIMP3 was used to treat the cancer cells at 25 MOI before the cells were implanted into the right flank of the mice. When the tumors can be touched, they were measured and recorded for the volumes. (a) The tumor growth curve. Ad-TIMP3-treated CT26 cells formed tumors were much smaller than that of the control group. (b) At the end of the observation, by tumor weight, Ad-TIMP3 pretreatment suppressed the tumor-formation ability of CT26 cells. (compared with Ad-Null, *Po0.05; #Po0.01). (c) Tumor growth curve during the Ad-TIMP3 treatment. Ad-TIMP3 significantly delayed the growth of tumors (Ad-TIMP3 compared with Ad-Null, *Po0.01, #Po0.05); (d) Compared with control Ad-Null, the tumor weight in Ad-TIMP3 was much lower than that in control groups (compared with Ad-con, *Po0.01, #Po0.05 ). (e) Metastatic lesions formed in liver. Eighteen mice were randomized into three groups and injected with variously treated CT26 cells (1 105 cells each). Seven days later, mice were killed and the liver metastatic lesions were counted on the sections (H&E staining). The Ad-TIMP3 group has much fewer lesions than other groups.

Article Snippet: The TIMP3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Infection, Tube Formation Assay, Control, Injection, Staining

Journal: Frontiers in Endocrinology

Article Title: MicroRNA-574 Impacts Granulosa Cell Estradiol Production via Targeting TIMP3 and ERK1/2 Signaling Pathway

doi: 10.3389/fendo.2022.852127

Figure Lengend Snippet: The expression of miR-574, pERK1/2 and TIMP3, during the antral follicular maturation. (A) The quantitative real-time PCR analysis of mature miR-574 in granulosa cells (GC) isolated from small-size (1-3 mm) and large-size (4-6 mm) follicles. (B) The quantitative real-time PCR analysis of miR-574 precursor in LGCs and SGCs. (C) The quantitative real-time PCR analysis of miR-574 in the follicular fluid (FF) isolated from small (S-FF) and large (L-FF) follicles. (D) Western blot analysis image showing increase pERK1/2 in LGC compared to SGC. (E) The quantitative real-time PCR analysis of TIMP3 mRNA in granulosa cells (GC) isolated from small- and large follicles. (F) Western blot analysis image showing increase TIMP3 in LGC compared to SGC. ns, no significant difference. Error bars represent means ± SD of at least three biological replicates and Asterisks denote a significant difference between groups (P<0.05) as determined by Student’s t -test.

Article Snippet: The primary antibodies include rabbit polyclonal ERK2 (K-23) antibodies (Cat.SC153; Santa Cruz Biotechnology, Santa Cruz, CA), anti-phosphoERK (Thr202/Tyr204) antibodies (Cat.#9101; Cell Signaling Technology, Danvers, MA), TIMP3 (Catalog Number: orb11485; Biorbyt), followed by incubation with secondary antibody anti-rabbit IgG horseradish peroxidase (Cat.#7074; 1:2000; Cell Signaling Technology) for at least one hour under room temperature.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Western Blot

Journal: Frontiers in Endocrinology

Article Title: MicroRNA-574 Impacts Granulosa Cell Estradiol Production via Targeting TIMP3 and ERK1/2 Signaling Pathway

doi: 10.3389/fendo.2022.852127

Figure Lengend Snippet: The primers used for Real-time qPCR.

Article Snippet: The primary antibodies include rabbit polyclonal ERK2 (K-23) antibodies (Cat.SC153; Santa Cruz Biotechnology, Santa Cruz, CA), anti-phosphoERK (Thr202/Tyr204) antibodies (Cat.#9101; Cell Signaling Technology, Danvers, MA), TIMP3 (Catalog Number: orb11485; Biorbyt), followed by incubation with secondary antibody anti-rabbit IgG horseradish peroxidase (Cat.#7074; 1:2000; Cell Signaling Technology) for at least one hour under room temperature.

Techniques: Sequencing

Journal: Frontiers in Endocrinology

Article Title: MicroRNA-574 Impacts Granulosa Cell Estradiol Production via Targeting TIMP3 and ERK1/2 Signaling Pathway

doi: 10.3389/fendo.2022.852127

Figure Lengend Snippet: Identification of putative miR-574 binding sites within the TIMP3 3-UTR. (A) Putative miR-574 binding sites at the 3’UTR of TIMP3 were computationally identified by the online target detection program ( https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid ); Top lines: miR-574 sequence; middle line: wild-type 3-UTR sequences; bottom line: mutated 3’UTR sequences. asterisks indicate the mutated nucleotides; (B) Granulosa cells were first transduced with lenti-miR-574 and then transfected with the luciferase-TIMP3 3-UTR vector or a control vector (a construct in which the 3-UTR of TIMP3 was replaced with a random sequence without any miR-574 target sites), and luciferase activity was measured. Mut represents luciferase expression constructs in which the miR-574 binding sites in the luciferase-TIMP3 3-UTR vector were mutated as indicated in (A) . The mean ± SEM of the relative luciferase expression ratio (Firefly luciferase/Rinella luciferase, Luc/R-luc) was calculated based on three biological replicates, and compared with the negative control (NC). Data represent the mean ± SE of three independent experiments. Asterisks denote statistically significant differences when compared to RFP (control; p <0.05).

Article Snippet: The primary antibodies include rabbit polyclonal ERK2 (K-23) antibodies (Cat.SC153; Santa Cruz Biotechnology, Santa Cruz, CA), anti-phosphoERK (Thr202/Tyr204) antibodies (Cat.#9101; Cell Signaling Technology, Danvers, MA), TIMP3 (Catalog Number: orb11485; Biorbyt), followed by incubation with secondary antibody anti-rabbit IgG horseradish peroxidase (Cat.#7074; 1:2000; Cell Signaling Technology) for at least one hour under room temperature.

Techniques: Binding Assay, Sequencing, Transduction, Transfection, Luciferase, Plasmid Preparation, Control, Construct, Activity Assay, Expressing, Negative Control

Journal: Frontiers in Endocrinology

Article Title: MicroRNA-574 Impacts Granulosa Cell Estradiol Production via Targeting TIMP3 and ERK1/2 Signaling Pathway

doi: 10.3389/fendo.2022.852127

Figure Lengend Snippet: miR-574 regulates TIMP3 in the cultured granulosa cell. (A) Relative levels of TIMP3 mRNA in the granulosa cell after overexpression of miR-574. (B) Representative western blot of TIMP3 protein in the granulosa cell after lentivirus transduction. (C) Densitometric quantitation depicting decreased expression of TIMP3 protein after over-expression of miR-574. GAPDH was used for western blot and mRNA normalization. The data represents the mean ± SE of three or four independent experiments. Asterisks denote statistically significant differences between the negative control (RFP) and miR-574 group (p <0.05). (D) Relative levels of TIMP3 mRNA after transfection with specific siRNA for miR-574 (miR-574 siRNA) and negative control siRNA (NC) in the granulosa cells. (E) Representative western blot image of TIMP3 protein after siRNA of miR-574 transfection. (F) Densitometric quantitation depicting increased expression of TIMP3 protein. (G) Relative levels of Aromatase mRNA in the granulosa cell after overexpression or knock-down of miR-574. (H) Relative levels of Cyp450scc mRNA in the granulosa cell after overexpression and knock-down of miR-574. GAPDH mRNA and protein were used for western blot and real-time qPCR normalization, respectively. The data represents the mean ± SE of three independent experiments. Asterisks denote statistically significant differences between NC and miR-574-siRNA groups (p <0.05). NS, not significant.

Article Snippet: The primary antibodies include rabbit polyclonal ERK2 (K-23) antibodies (Cat.SC153; Santa Cruz Biotechnology, Santa Cruz, CA), anti-phosphoERK (Thr202/Tyr204) antibodies (Cat.#9101; Cell Signaling Technology, Danvers, MA), TIMP3 (Catalog Number: orb11485; Biorbyt), followed by incubation with secondary antibody anti-rabbit IgG horseradish peroxidase (Cat.#7074; 1:2000; Cell Signaling Technology) for at least one hour under room temperature.

Techniques: Cell Culture, Over Expression, Western Blot, Transduction, Quantitation Assay, Expressing, Negative Control, Transfection, Knockdown

Journal: Frontiers in Endocrinology

Article Title: MicroRNA-574 Impacts Granulosa Cell Estradiol Production via Targeting TIMP3 and ERK1/2 Signaling Pathway

doi: 10.3389/fendo.2022.852127

Figure Lengend Snippet: Inhibition of TIMP3 decreased phosphorylated ERK1/2 but increased estradiol production in cultured granulosa cells. (A) Relative levels of TIMP3 mRNA after transfection with siRNA for TIMP3 (si-TIMP3) and negative control siRNA (NC) in granulosa cells. (B) Representative western blot of TIMP3 in granulosa cells after transfection with siRNA for TIMP3 (si-TIMP3) and negative control siRNA (NC). (C) Representative western blot of pERK1/2 and ERK1/2 protein after siRNA transfection. (D) Densitometric quantitation depicting decreased level of pERK1/2. (E) Suppression of TIMP3 promoted granulosa cells to produce more estradiol. GAPDH protein and mRNA were used for western blot data and mRNA normalization. Estradiol production was measured by ELISA assay. Data represent the mean ± SE of three independent experiments. Asterisks denote statistically significant differences between NC and si-TIMP3 group (p <0.05).

Article Snippet: The primary antibodies include rabbit polyclonal ERK2 (K-23) antibodies (Cat.SC153; Santa Cruz Biotechnology, Santa Cruz, CA), anti-phosphoERK (Thr202/Tyr204) antibodies (Cat.#9101; Cell Signaling Technology, Danvers, MA), TIMP3 (Catalog Number: orb11485; Biorbyt), followed by incubation with secondary antibody anti-rabbit IgG horseradish peroxidase (Cat.#7074; 1:2000; Cell Signaling Technology) for at least one hour under room temperature.

Techniques: Inhibition, Cell Culture, Transfection, Negative Control, Western Blot, Quantitation Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Endocrinology

Article Title: MicroRNA-574 Impacts Granulosa Cell Estradiol Production via Targeting TIMP3 and ERK1/2 Signaling Pathway

doi: 10.3389/fendo.2022.852127

Figure Lengend Snippet: Recombinant TIMP3 increased phosphorylated ERK1/2 level and decreased estradiol production. (A) The PathDetect in vivo signal transduction pathway trans-reporting systems (Promega) was used to measure transcription activator and ERK1/2 signal transduction pathway after the addition of Recombinant TIMP3. First, the cultured granulosa cells were co-transfected with a pFR-luciferase reporter with a pFA2-Elk1 plasmid. 24h after the transfection, the recombinant TIMP3(rTIMP3) was added into the culture plate well. Granulosa cell sample was collected after 6h treatment then used for luciferase assay. Luciferase activity is indicative of transcription factor-dependent activation, is expressed as relative light units compared to control. (B) Estradiol production was measured by ELISA assay. The addition of TIMP3 suppresses estradiol production in the cultured granulosa cells. The data represents the mean ± SE of three or four independent experiments. Asterisks denote statistically significant differences between the blank control and the rTIMP3 group (p <0.05).

Article Snippet: The primary antibodies include rabbit polyclonal ERK2 (K-23) antibodies (Cat.SC153; Santa Cruz Biotechnology, Santa Cruz, CA), anti-phosphoERK (Thr202/Tyr204) antibodies (Cat.#9101; Cell Signaling Technology, Danvers, MA), TIMP3 (Catalog Number: orb11485; Biorbyt), followed by incubation with secondary antibody anti-rabbit IgG horseradish peroxidase (Cat.#7074; 1:2000; Cell Signaling Technology) for at least one hour under room temperature.

Techniques: Recombinant, In Vivo, Transduction, Cell Culture, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Activation Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: Diagnostic Pathology

Article Title: Promoter methylation and expression of TIMP3 gene in gastric cancer

doi: 10.1186/1746-1596-8-110

Figure Lengend Snippet: Gastric carcinoma TIMP3 promoter methylation and protein expression

Article Snippet: TIMP3 antibody and SP kit were purchased from Boster Biological Engineering Co., Ltd. (Dalian).

Techniques: Methylation, Expressing

Journal: Diagnostic Pathology

Article Title: Promoter methylation and expression of TIMP3 gene in gastric cancer

doi: 10.1186/1746-1596-8-110

Figure Lengend Snippet: TIMP3 protein immunohistochemisty (SP 400×): a, normal gastric tissue; b, early gastric cancer; c, advanced gastric cancer; d, transfer of lymph node.

Article Snippet: TIMP3 antibody and SP kit were purchased from Boster Biological Engineering Co., Ltd. (Dalian).

Techniques:

Journal: Diagnostic Pathology

Article Title: Promoter methylation and expression of TIMP3 gene in gastric cancer

doi: 10.1186/1746-1596-8-110

Figure Lengend Snippet: Relationship between advanced gastric cancer pathology TIMP3 methylation and its protein expression level

Article Snippet: TIMP3 antibody and SP kit were purchased from Boster Biological Engineering Co., Ltd. (Dalian).

Techniques: Methylation, Expressing