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