Sb431542

To evaluate the effect of TGF-β1 among MSC-secreted soluble factors, anti-TGF-β1 antibodies (ab64715, Abcam, United Kingdom) of 10 μg/ml were added into the medium of indirect MSC-treated CD4⁺ T cell group before LPS (100 ng/ml) and hypoxia (5% O₂) stimulation and then followed by Treg/Th17 and miR-155 expression evaluation.
In addition, ALK5 inhibitor was used for blocking the TGF-β-mediated signaling. In the case of ALK5 inhibitor treatment (SB431542, Sigma, Taufkirchen, Germany), cells were preincubated with 1 μm SB-431542 dissolved in DMSO for 30 min on ice before putting the cells into the coculture system with MSCs receiving LPS (100 ng/ml) and hypoxia (5% O₂) stimulation and then followed by Treg/Th17 detection.

To initiate the differentiation of mouse iPSCs to ECs, medium was changed to RPMI-1640 containing 2% B-27 minus insulin (both Gibco) and 5 μM CHIR-99021 (Selleckchem). Starting on day 2, RPMI-1640 containing 2% B-27 minus insulin (both Gibco) and 2 μM CHIR-99021 (Selleckchem) was used. From day 4 to day 7, cells were exposed to RPMI-1640 EC medium containing 2% B-27 minus insulin plus 50 ng ml⁻¹ of mouse vascular endothelial growth factor (mVEGF, R&D Systems), 10 ng ml⁻¹ of mouse fibroblast growth factor basic (mFGFb, R&D Systems), 10 μM Y-27632 (Sigma-Aldrich) and 1 μM SB 431542 (Sigma-Aldrich). EC clusters were visible from day 7, and cells were maintained in Endothelial Cell Basal Medium 2 (PromoCell) plus supplements, 10% FCS heat-inactivated (HI) (Gibco), 1% penicillin–streptomycin, 25 ng ml⁻¹ of VEGF, 2 ng ml⁻¹ of FGFb, 10 μM Y-27632 (Sigma-Aldrich) and 1 μM SB 431542 (Sigma-Aldrich). The differentiation process was completed after 21 days, and undifferentiated cells were detached during the differentiation process. For purification, cells went through MACS purification using anti-CD15 mAb-coated magnetic microbeads (Miltenyi) for negative selection.

RCC cells were seeded in 100 mm cell culture dishes 24 h prior stimulation. For TGF-β1 stimulation, the cells were grown for 96 h in the presence of 10 ng/mL TGF-β1 (Biolegend, San Diego, CA, USA, Cat. No. 580704). For TGFBR1 inhibition, the cells were treated with 10 ng/mL SB 431542 (Sigma, St. Louis, MO, USA, CAS no. 301836-41-9) for 96 h. To refresh the stimulus or inhibitor, the medium was changed once after 48 h. For re-culture experiments, the cells were shifted to medium without TGF-β1 or DMEM supplemented with 10 ng/mL SB 431542 for 96 h after 96 h TGF-β1 treatment. Analogous, the medium was refreshed after 48 h. Untreated cells grown for the same period of time served as unstimulated controls.

CXCR4, CXCL12, and SB431542 were purchased from Sigma (St. Louis, MO, USA) and the neutralizing Abs for CXCR4 and CXCL12 were purchased from R&D Systems Inc. (Minneapolis, MN, USA). To study TGF-β signaling, a small molecular inhibitor (SB431542, Sigma) was used.

Mouse C2C12 myoblasts were grown to confluency in 6-well plates in proliferating media consisting of Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). At confluency, the media was removed and cells were washed twice in 1x phosphate buffered saline (PBS) and covered with differentiating media consisting of DMEM supplemented with 2% horse serum and 1% P/S. Cells were harvested at 1 day (1 D) and 3 days (3 D) post-induction of differentiation. One day prior to cell harvest, media was supplemented with vehicle (4 mM HCl with 0.1% BSA), recombinant myostatin (R&D Laboratories) at 100 ng/ml and 1 µg/ml without or without co-incubation with 1 µM SB431542 (Sigma, St. Louis, MO USA) and 1 µM SB431542 alone. SB431542 is a small molecule that selectively inhibits ALK 4, 5 and 7 [27] ; at 1 µM SB431542 has been shown to significantly reduce Smad2 phosphorylation in C2C12 cells [19] (link).