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: International Journal of Molecular Sciences

Article Title: Betaglycan (TβRIII) is a Key Factor in TGF-β2 Signaling in Prepubertal Rat Sertoli Cells

doi: 10.3390/ijms20246214

Figure Lengend Snippet: TGF-β2 treatment induces TIMP3 mRNA and secretion. The 1 × 10 5 93RS2 cells/well were incubated with TGF-β1 or TGF-β2 (both 10 ng/mL) for ( A ) 24 h or ( B ) 48 h and the mRNA expression of TIMP3 measured with qRT-PCR. Only TGF-β2 stimulated TIMP3 mRNA expression significantly given as fold change of control. 1 × 10 5 93RS2 cells/well were incubated with TGF-β2 for ( C ) 24 h or ( D ) 48 h. Supernatants were analyzed for TIMP3 by ELISA. TGF-β2 stimulated secretion of TIMP3 dose-dependently and significantly. Each bar represents the mean ± SEM of 3 independent experiments performed in duplicate. Dunnett’s test was used for statistical analysis; ** p < 0.01, *** p < 0.001, ns = not significant.

Article Snippet: We used the following materials and kits: recombinant human TIMP3 (R&D Systems, Wiesbaden, Germany) and recombinant human TGF-β1 and TGF-β2 (Promokine, Heidelberg Germany), protease inhibitor cocktail, Fluoroprofile protein quantification kit (Merck/Sigma Aldrich, Darmstadt, Germany), siRNAs (Thermo Fisher/Invitrogen, Carlsbad, CA, USA) and GM6001 (broad-spectrum inhibitor of MMPs; Sigma Aldrich, St. Louis, Missouri, USA), TGF-β1 (range 31.2–2000 pg/mL, sensitivity 15.6 pg/mL, cat-no MB100B), TGF-β2 (range 31.2–2000 pg/mL, sensitivity 15.6 pg/mL, cat-no DY302), and TIMP3 (range 62.5–4000 pg/mL, cat-no DY973) ELISAs (all from R&D Systems, Wiesbaden, Germany), TIMP1 (range 0.156–10 ng/mL, sensitivity 0.05 ng/mL, cat-no MBS263032), TIMP2 (range 78–5000pg/mL, sensitivity 39 pg/mL, cat-no MBS2880823), rat TβRIII/sBG ELISA (range 0.156–10 ng/mL, sensitivity 0.086 ng/mL; cat-no MBS289506, all from MyBioSource, San Diego, CA, USA), rat P-Smad3 ELISA (range 10–500 μg/mL, sensitivity 10 μg/mL; cat-no ab186038, Abcam, Cambridge, UK) and P-Smad2 ELISA (10–1000 μg/mL; Cell Signaling, cat-no 7384c, Frankfurt, Germany).

Techniques: Incubation, Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Betaglycan (TβRIII) is a Key Factor in TGF-β2 Signaling in Prepubertal Rat Sertoli Cells

doi: 10.3390/ijms20246214

Figure Lengend Snippet: TIMP3 treatment reduces secretion of TGF-β1, TGF-β2 and shedding of BG. The 1 × 10 5 93RS2 cells/well were incubated with TIMP3 for 48 h. Supernatants were analyzed for TGF-β1 ( A ), TGF-β2 ( B ) and sBG ( C ) by ELISA. TIMP3 reduced secretion of TGF-β1 ( A ), TGF-β2 ( B ) and shedding of sBG ( C ) dose-dependently and significantly. Each bar represents the mean ± SEM of 3 independent experiments performed in duplicate. Dunnett’s test was used for statistical analysis; * p ≤ 0.05, ** p < 0.01, *** p < 0.001, ns = not significant, rhTIMP3 = recombinant TIMP3, ns = not significant.

Article Snippet: We used the following materials and kits: recombinant human TIMP3 (R&D Systems, Wiesbaden, Germany) and recombinant human TGF-β1 and TGF-β2 (Promokine, Heidelberg Germany), protease inhibitor cocktail, Fluoroprofile protein quantification kit (Merck/Sigma Aldrich, Darmstadt, Germany), siRNAs (Thermo Fisher/Invitrogen, Carlsbad, CA, USA) and GM6001 (broad-spectrum inhibitor of MMPs; Sigma Aldrich, St. Louis, Missouri, USA), TGF-β1 (range 31.2–2000 pg/mL, sensitivity 15.6 pg/mL, cat-no MB100B), TGF-β2 (range 31.2–2000 pg/mL, sensitivity 15.6 pg/mL, cat-no DY302), and TIMP3 (range 62.5–4000 pg/mL, cat-no DY973) ELISAs (all from R&D Systems, Wiesbaden, Germany), TIMP1 (range 0.156–10 ng/mL, sensitivity 0.05 ng/mL, cat-no MBS263032), TIMP2 (range 78–5000pg/mL, sensitivity 39 pg/mL, cat-no MBS2880823), rat TβRIII/sBG ELISA (range 0.156–10 ng/mL, sensitivity 0.086 ng/mL; cat-no MBS289506, all from MyBioSource, San Diego, CA, USA), rat P-Smad3 ELISA (range 10–500 μg/mL, sensitivity 10 μg/mL; cat-no ab186038, Abcam, Cambridge, UK) and P-Smad2 ELISA (10–1000 μg/mL; Cell Signaling, cat-no 7384c, Frankfurt, Germany).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: Betaglycan (TβRIII) is a Key Factor in TGF-β2 Signaling in Prepubertal Rat Sertoli Cells

doi: 10.3390/ijms20246214

Figure Lengend Snippet: Effects of TIMP3 on Smad3 phosphorylation in TGF-β1/2 signaling. The 7 × 10 4 93RS2 cells/well were incubated with different doses of TIMP3 for 2 h and then treated with ( A ) TGF-β1 or ( B ) TGF-β2 (both 10 ng/mL) for 2 h. Cell lysates were analyzed for p-Smad3 by ELISA. TIMP3 only increased phosphorylation of Smad3 together with TGF-β2 ( B ) dose-dependently and significantly. Each bar represents the mean ± SEM of 3 independent experiments performed in duplicate. Dunnett’s test was used for statistical analysis; ** p < 0.01; *** p < 0.001, ns = not significant.

Article Snippet: We used the following materials and kits: recombinant human TIMP3 (R&D Systems, Wiesbaden, Germany) and recombinant human TGF-β1 and TGF-β2 (Promokine, Heidelberg Germany), protease inhibitor cocktail, Fluoroprofile protein quantification kit (Merck/Sigma Aldrich, Darmstadt, Germany), siRNAs (Thermo Fisher/Invitrogen, Carlsbad, CA, USA) and GM6001 (broad-spectrum inhibitor of MMPs; Sigma Aldrich, St. Louis, Missouri, USA), TGF-β1 (range 31.2–2000 pg/mL, sensitivity 15.6 pg/mL, cat-no MB100B), TGF-β2 (range 31.2–2000 pg/mL, sensitivity 15.6 pg/mL, cat-no DY302), and TIMP3 (range 62.5–4000 pg/mL, cat-no DY973) ELISAs (all from R&D Systems, Wiesbaden, Germany), TIMP1 (range 0.156–10 ng/mL, sensitivity 0.05 ng/mL, cat-no MBS263032), TIMP2 (range 78–5000pg/mL, sensitivity 39 pg/mL, cat-no MBS2880823), rat TβRIII/sBG ELISA (range 0.156–10 ng/mL, sensitivity 0.086 ng/mL; cat-no MBS289506, all from MyBioSource, San Diego, CA, USA), rat P-Smad3 ELISA (range 10–500 μg/mL, sensitivity 10 μg/mL; cat-no ab186038, Abcam, Cambridge, UK) and P-Smad2 ELISA (10–1000 μg/mL; Cell Signaling, cat-no 7384c, Frankfurt, Germany).

Techniques: Phospho-proteomics, Incubation, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Betaglycan (TβRIII) is a Key Factor in TGF-β2 Signaling in Prepubertal Rat Sertoli Cells

doi: 10.3390/ijms20246214

Figure Lengend Snippet: Effects of TIMP3 silencing on Smad2/3 phosphorylation in TGF-β1/2 signaling. A control RT-PCR of the knockdown effectiveness for TIMP3 was performed to ensure silencing of TIMP3 ( A ). The 7 × 10 4 93RS2 TIMP3-silenced cells were treated with TGF-β1 or TGF-β2 (both 10 ng/mL) for 1 h. Cell lysates were analyzed for P-Smad2 and P-Smad3 by ELISAs. Silencing of TIMP3 attenuated TGF-β2 but not TGF-β1-dependent phosphorylation of Smad2 ( B ) and of Smad3 ( C ) as well as proliferation of 93RS2 cells by 50% ( D ). Each bar represents the mean ± SEM of 3 independent experiments performed in duplicate. Dunnett’s test was used for statistical analysis; * p ≤ 0.05, ** p < 0.01; *** p < 0.001; Ctrl, control; nc-siRNA, negative control siRNA; M, Marker; +, addition of substance; -, without addition of substance.

Article Snippet: We used the following materials and kits: recombinant human TIMP3 (R&D Systems, Wiesbaden, Germany) and recombinant human TGF-β1 and TGF-β2 (Promokine, Heidelberg Germany), protease inhibitor cocktail, Fluoroprofile protein quantification kit (Merck/Sigma Aldrich, Darmstadt, Germany), siRNAs (Thermo Fisher/Invitrogen, Carlsbad, CA, USA) and GM6001 (broad-spectrum inhibitor of MMPs; Sigma Aldrich, St. Louis, Missouri, USA), TGF-β1 (range 31.2–2000 pg/mL, sensitivity 15.6 pg/mL, cat-no MB100B), TGF-β2 (range 31.2–2000 pg/mL, sensitivity 15.6 pg/mL, cat-no DY302), and TIMP3 (range 62.5–4000 pg/mL, cat-no DY973) ELISAs (all from R&D Systems, Wiesbaden, Germany), TIMP1 (range 0.156–10 ng/mL, sensitivity 0.05 ng/mL, cat-no MBS263032), TIMP2 (range 78–5000pg/mL, sensitivity 39 pg/mL, cat-no MBS2880823), rat TβRIII/sBG ELISA (range 0.156–10 ng/mL, sensitivity 0.086 ng/mL; cat-no MBS289506, all from MyBioSource, San Diego, CA, USA), rat P-Smad3 ELISA (range 10–500 μg/mL, sensitivity 10 μg/mL; cat-no ab186038, Abcam, Cambridge, UK) and P-Smad2 ELISA (10–1000 μg/mL; Cell Signaling, cat-no 7384c, Frankfurt, Germany).

Techniques: Phospho-proteomics, Control, Reverse Transcription Polymerase Chain Reaction, Knockdown, Negative Control, Marker

Journal: International Journal of Molecular Sciences

Article Title: Betaglycan (TβRIII) is a Key Factor in TGF-β2 Signaling in Prepubertal Rat Sertoli Cells

doi: 10.3390/ijms20246214

Figure Lengend Snippet: Scheme for the different modes of signaling by TGF-β1 and TGF-β2 under the influence of BG and TIMP3. ( A ) Normally binding of TGF-β1 (b1) to the TGF-β receptor complex results in phosphorylation of Smad3. However, TIMP3 counteracts MMP-mediated BG shedding, thus resulting in interference of membrane-bound and ligand-activated BG with the TGF-β receptor complex. This interaction attenuates TGF-β1-dependent Smad3 phosphorylation via TβRI. sBG reduces binding of TGF-β1 to the TGF-β receptor complex and thus counteracts TGF-β1 signaling. ( B ) In contrast, binding of TGF-β2 (b2) to BG enhances phosphorylation of TGF-β2-dependent Smad3 which is further increased when TIMP3 inhibits MMP-dependent BG shedding. sBG reduces binding of TGF-β2 to the TGF-β receptor complex and thus counteracts TGF-β2 signaling.

Article Snippet: We used the following materials and kits: recombinant human TIMP3 (R&D Systems, Wiesbaden, Germany) and recombinant human TGF-β1 and TGF-β2 (Promokine, Heidelberg Germany), protease inhibitor cocktail, Fluoroprofile protein quantification kit (Merck/Sigma Aldrich, Darmstadt, Germany), siRNAs (Thermo Fisher/Invitrogen, Carlsbad, CA, USA) and GM6001 (broad-spectrum inhibitor of MMPs; Sigma Aldrich, St. Louis, Missouri, USA), TGF-β1 (range 31.2–2000 pg/mL, sensitivity 15.6 pg/mL, cat-no MB100B), TGF-β2 (range 31.2–2000 pg/mL, sensitivity 15.6 pg/mL, cat-no DY302), and TIMP3 (range 62.5–4000 pg/mL, cat-no DY973) ELISAs (all from R&D Systems, Wiesbaden, Germany), TIMP1 (range 0.156–10 ng/mL, sensitivity 0.05 ng/mL, cat-no MBS263032), TIMP2 (range 78–5000pg/mL, sensitivity 39 pg/mL, cat-no MBS2880823), rat TβRIII/sBG ELISA (range 0.156–10 ng/mL, sensitivity 0.086 ng/mL; cat-no MBS289506, all from MyBioSource, San Diego, CA, USA), rat P-Smad3 ELISA (range 10–500 μg/mL, sensitivity 10 μg/mL; cat-no ab186038, Abcam, Cambridge, UK) and P-Smad2 ELISA (10–1000 μg/mL; Cell Signaling, cat-no 7384c, Frankfurt, Germany).

Techniques: Binding Assay, Phospho-proteomics, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Betaglycan (TβRIII) is a Key Factor in TGF-β2 Signaling in Prepubertal Rat Sertoli Cells

doi: 10.3390/ijms20246214

Figure Lengend Snippet: List of primer sequences used for RT-PCR.

Article Snippet: We used the following materials and kits: recombinant human TIMP3 (R&D Systems, Wiesbaden, Germany) and recombinant human TGF-β1 and TGF-β2 (Promokine, Heidelberg Germany), protease inhibitor cocktail, Fluoroprofile protein quantification kit (Merck/Sigma Aldrich, Darmstadt, Germany), siRNAs (Thermo Fisher/Invitrogen, Carlsbad, CA, USA) and GM6001 (broad-spectrum inhibitor of MMPs; Sigma Aldrich, St. Louis, Missouri, USA), TGF-β1 (range 31.2–2000 pg/mL, sensitivity 15.6 pg/mL, cat-no MB100B), TGF-β2 (range 31.2–2000 pg/mL, sensitivity 15.6 pg/mL, cat-no DY302), and TIMP3 (range 62.5–4000 pg/mL, cat-no DY973) ELISAs (all from R&D Systems, Wiesbaden, Germany), TIMP1 (range 0.156–10 ng/mL, sensitivity 0.05 ng/mL, cat-no MBS263032), TIMP2 (range 78–5000pg/mL, sensitivity 39 pg/mL, cat-no MBS2880823), rat TβRIII/sBG ELISA (range 0.156–10 ng/mL, sensitivity 0.086 ng/mL; cat-no MBS289506, all from MyBioSource, San Diego, CA, USA), rat P-Smad3 ELISA (range 10–500 μg/mL, sensitivity 10 μg/mL; cat-no ab186038, Abcam, Cambridge, UK) and P-Smad2 ELISA (10–1000 μg/mL; Cell Signaling, cat-no 7384c, Frankfurt, Germany).

Techniques: Sequencing