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: 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

Journal: Cancers

Article Title: Fibroblasts in Nodular Sclerosing Classical Hodgkin Lymphoma Are Defined by a Specific Phenotype and Protect Tumor Cells from Brentuximab-Vedotin Induced Injury

doi: 10.3390/cancers11111687

Figure Lengend Snippet: Fibroblasts derived from nodular sclerosing subtype of cHL (NS cHL) and lymphadenitis (LA) show considerable differences in their gene expression program. ( A ) Unsupervised hierarchical gene expression clustering of fibroblast samples derived from lymphadenitis (Fib LA, yellow, n = 5), from mixed cellularity subtype of cHL (MC cHL, blue, n = 5) and from NS cHL (red, n = 7) considering 185 transcripts with a standard deviation >1. ( B ) Principal component analysis considering the same 185 transcripts with a standard deviation >1. Fibroblasts from LA yellow, MC cHL blue) and from NS cHL (red). ( C ) Quantitative real time PCR showing significantly higher myocardin ( MYOCD ) transcript levels in MC cHL ( n = 5) and NS cHL ( n = 8) compared with fibroblasts from lymphadenitis ( n = 5) (Mann–Whitney test, ** p < 0.01). ( D ) Quantitative real time PCR showing significantly higher tissue inhibitor of metalloproteinase 3 ( TIMP3 ) transcript levels in NS cHL ( n = 8) compared with fibroblasts from lymphadenitis ( n = 5) (Mann–Whitney test, ** p = 0.002). ( E ) Representative immunohistochemical TIMP3 staining of a lymphadenitis case (100×). TIMP3 is expressed in paraimmunoblasts (insert, 400×). ( F ) Representative immunohistochemical staining for TIMP3 of a NS cHL (100×) with expression of TIMP3 in fibroblasts (insert 1) and Hodgkin- and Reed-Sternberg (HRS) cells (insert 2).

Article Snippet: Furthermore, TIMP3 secretion was analyzed using Human TIMP3 PicoKine ELISA Kit according to manufacturer´s instruction (BosterBio, Pleasanton, CA, USA).

Techniques: Derivative Assay, Expressing, Standard Deviation, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Immunohistochemical staining, Staining

Journal: Cancers

Article Title: Fibroblasts in Nodular Sclerosing Classical Hodgkin Lymphoma Are Defined by a Specific Phenotype and Protect Tumor Cells from Brentuximab-Vedotin Induced Injury

doi: 10.3390/cancers11111687

Figure Lengend Snippet: Genes differentially expressed between fibroblasts derived from cHL and lymphadenitis.

Article Snippet: Furthermore, TIMP3 secretion was analyzed using Human TIMP3 PicoKine ELISA Kit according to manufacturer´s instruction (BosterBio, Pleasanton, CA, USA).

Techniques: Derivative Assay, Binding Assay

Journal: Cancers

Article Title: Fibroblasts in Nodular Sclerosing Classical Hodgkin Lymphoma Are Defined by a Specific Phenotype and Protect Tumor Cells from Brentuximab-Vedotin Induced Injury

doi: 10.3390/cancers11111687

Figure Lengend Snippet: NS cHL fibroblasts show enhanced proliferation in the presence of conditioned medium derived from cHL cell lines, even after application of luteolin. ( A ) NS cHL fibroblasts (CAF) show a significantly higher proliferation after the application of conditioned media from the cHL cell lines L-428 and L-1236 when compared with standard (IMDM) or mock cell culture medium (RPMI 1640 medium) only orconditioned medium obtained from the Burkitt lymphoma cell line Raji or IL-7. Proliferation was measured by Cell Trace Violet Blue. Fluorescence values were inverted and normalized to the control (CAF). Mean + SEM of three independent experiments in triplicates, ** p < 0.01, *** p < 0.001, One-Way ANOVA with Bonferroni correction for multiple comparisons. ( B ) NS cHL fibroblasts (CAF) show a significantly decreased proliferation after the application of luteolin, which can be reversed by addition of conditioned media from the cHL cell lines L-428 and L-1236, but not by addition of IL-7. Proliferation was measured by Cell Trace Violet Blue. Fluorescence values were inverted and normalized to the control (CAF). Mean + SEM of three independent experiments in triplicates,* p < 0.05, Kruskal–Wallis test with Dunn’s correction for multiple comparisons. ( C ) Viable cells in untreated and luteolin treated fibroblasts 48 h after luteolin treatment. *** p < 0.001 unpaired t -test. Four experiments with three replicates. ( D ) Absorption at 450 nm is significantly decreased in the supernatants derived from luteolin treated fibroblasts after 48 h reflecting a significantly downregulated TIMP3 secretion in the ELISA. Mean + SEM of four independent experiments in triplicates. *** p < 0.001, paired t -test.

Article Snippet: Furthermore, TIMP3 secretion was analyzed using Human TIMP3 PicoKine ELISA Kit according to manufacturer´s instruction (BosterBio, Pleasanton, CA, USA).

Techniques: Derivative Assay, Cell Culture, Fluorescence, Enzyme-linked Immunosorbent Assay