Tgf β

CH12F3 cell lines were stimulated with anti‐CD40 (16‐0402‐86; ebioscience; RRID: AB_468947), IL4 (CK15; Novoprotein) plus TGF‐β (CA59; Novoprotein). CSR to IgA was monitored at the indicated time. Splenic naïve B cells were purified as previously described (Qiao et al, 2017 (link)) and cultured in RPMI1640 plus 15% fetal bovine serum (FBS, ExCell Bio, catalog # FSP500, Lot No.11H235). LPS (sigma; L2630‐100MG) and IL4 (CK15; Novoprotein) were used to activate splenic naïve B cells. Infected with retrovirus on Day 1; CSR to IgG1 was monitored at Day 3 and Day 4. For retroviral AID expression, the Aicda gene and its mutants were cloned into a pMX‐IRES‐GFP or pMX‐IRES‐Puro vector (Basu et al, 2008 (link)). The plasmid was co‐transfected with a packaging plasmid pCL‐10A1 (Novus Biologicals) into HEK293T cells using Ca₃(PO₄)₂ co‐precipitation approach. Cell proliferation was monitored by cell counting with a hemocytometer. The cell viability of retroviral vector‐infected B cells was calculated by the fraction of GFP⁺ cells in the whole population.

Human foreskin fibroblast (HFF) and human embryonic kidney cells (HEK 293T) were cultured in Dulbecco’s modified Eagle medium (DMEM) (GIBCO, ThermoFisher, Waltham, MA, United States) supplemented with 10% pre-selected fetal bovine serum (FBS) (GIBCO, ThermoFisher, Waltham, MA, United States), 1% penicillin and streptomycin (GIBCO, ThermoFisher, Waltham, MA, United States) in a 37°C humidified incubator with 5% CO₂. Human umbilical cord mesenchymal stem cells (hUCMSCs) were cultured in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 HAM (1:1) (DMEM/F12, GIBCO, ThermoFisher, Waltham, MA, United States) containing 10% FBS and 1% penicillin/streptomycin in a 37°C humidified incubator with 5% CO₂.
NIH 3T3 cells were maintained in DMEM with 10% FBS and kept in a 37°C incubator with a 5% CO₂ air atmosphere, and TGF-β (Novoprotein, China) was added with 2 ng/ml. The total proteins of cells were harvested 24 h post-treatment.

Primary cardiac fibroblasts were transfected with H1.0 or scrambled negative control siRNA for 48 h. Fibroblasts suspended in 10% serum-supplemented DMEM/F-12 medium were seeded (0.5 × 10⁶ cells per milliliter) on collagen gels 24 h before serum deprivation for 4 h. At the beginning of contraction, gels were released from wells using a pipette tip and treated with TGF-β (10 ng ml⁻¹; Novoprotein, CA59) for 24 h. Primary cardiac fibroblasts transfected with Adv-GFP or Adv-GFP-H1.0-FLAG for 48 h were suspended in 10% serum-supplemented DMEM/F-12 medium, seeded (0.5 × 10⁶ cells per milliliter) on collagen gels for 8 h and then released from wells for 24 h. Gel images were acquired by the ChemiDoc MP Imaging System (Bio-Rad). Gel area was calculated using ImageJ^{73 (link)} and Fiji^{74 (link)}, and contraction was reported as percentage of contraction.

T lymphocytes were obtained from the spleen using the BioLegend Natural CD4 + T Cell Purification Kit (BioLegend) and then seeded on anti-CD3-coated plates at a density of 5 × 105 cells/well. CD4 + T cells and saline/MSCs/IL-1β-primed MSCs were cocultured at a ratio of 5:1. Treg cell differentiation was induced with RPMI 1640 containing 10% FBS, 3 μg/mL anti-CD28 (BioLegend), 50 ng/mL IL-2 (Novoprotein), and 5 ng/mL TGF-β (Novoprotein). After 72 h of cocultivation, CD4 + FOXP3 + Treg cells were collected and analyzed by flow cytometry.

Murine naïve CD4⁺T cells were purified from the spleen of male C57BL/6 mice by magnetic cell negative selection using a mouse naïve CD4⁺T cell isolation kit (Miltenyi Biotec, 130-104-453), according to manufacturer’s instructions. Cells were stimulated in vitro with plate-bound anti-CD3 (BD Pharmingen, 553057) and anti-CD28 antibodies (BD Pharmingen, 553294) (both for 2 μg/mL) in RPMI 1640, supplemented with 10% heat-inactivated fetal bovine serum and 100 U/mL penicillin/streptomycin. To induce the differentiation of Th17 cells, cells were activated in the presence of 25 ng/mL IL-6 (Novoprotein, CG39), 20 ng/mL IL-23 (Novoprotein, CI18), 2.5 ng/mL TGF-β (Novoprotein, CA59). PNS, Tepp-46 (Apexbio, C4181), and SAICAR (Apexbio, C3413) were dissolved at a concentration of 10 mM in dimethyl sulfoxide (DMSO) and stored at −20 °C until use. For PNS treatment, IL-6/IL-23/TGF-β were added to each well, followed by treatment with PNS (5, 10, 20 μg/mL) and incubation for 72 h. For Tepp-46 and SAICAR treatment, cells were pre-incubated at 37 °C for 24 h with Tepp-46 (50, 100, 150 μM) or SAICAR (2, 4, 8 μM) before IL-6/IL-23/TGF-β activation. For co-treatment, cells were pretreated with SAICAR (4 μM) or Tepp-46 (100 μM) for 24 h before the condition of Th17-polarization), and treated with PNS (10 μg/mL) for another 72 h.