Mir mimic

Human N-Ras 3′-UTR (3630 bp) containing the putative binding sites of miR98 and Lin28b 3′-UTR (4548 bp) with the putative binding sites of miR27b were amplified by PCR, inserted into the firefly luciferase reporter vector pmiR-RB-Reporter^™ (pWT, RiboBio Co. Ltd, China) between the restrictive sites Xho I and Not I, and validated by sequencing. Its mutant constructs (N-Ras or Lin28b 3′-UTR,pMut) with a mutation of the miRNA (miR98 or miR27b) binding site was generated with the mutagenic oligo nucleotide primers (Supplementary Table 1), according to the manual of GeneTailor Site Directed Mutagenesis System (Invitrogen). The 293T cells were transfected with miR-mimics (RiboBio Co. Ltd) and pWT, or miR-mimcs and pMut, or miR-negative control (nc) and pWT, or miR-nc and pMut. Cells were harvested after 24 hr and the luciferase activity was assayed according to the dual-luciferase assay manual (Promega, USA). The Renilla luciferase signal was normalized to the firefly luciferase signal for each individual analysis.

Rat kidney fibroblast cell NRK-49F was obtained from Millipore Sigma and was cultured in Dulbecco's modified Eagle’s medium (DMEM) supplemented with 10% FBS. Cells were treated with TGF-β1 (10 ng/mL) for 48 h to induce fibrosis according to our pilot experience [8 ]. To modulate miR-29c-3p expression levels, NRK-49F cells were transfected with miR mimic or inhibitor, both synthesized by Ribobio (Guangzhou, China). The Fer inhibitor DS21360717 was obtained from ChemSrc. Adenoviral over-expression was performed using a Fer cDNA clone obtained from Origene. The Fer knockdown (KD) modeling was constructed using shRNA, the sequence of which was obtained from TRC (TRCN0000361140). The treatment dose was designated at 5 nM for the interaction for 24 h before testing. All transfections were done using Lipofectamine 2000 system according to the manufacturer’s instructions. The cells were harvested 48 h after transfection for further studies.

Human peripheral blood (PB) leukapheresis products of volunteers were obtained from the Department of Hematology in the Affiliated Jiangning Hospital of Nanjing Medical University. Naive human PB T cells (CD4⁺CD45RA⁺) were sort purified from PB mononuclear cells (PBMCs) (Ficoll-Hypaque, Amersham Biosciences) by magnetic-activated cell sorting (MACS pro) (Miltenyi Biotec, Germany) in a two-step procedure of magnetic beads sorting.
Naive T cells were induced to iTregs with anti-CD3/CD28 mAb-coated Dynabeads (Life Technologies, Carlsbad, CA, USA) at 1:1 (cell-to-bead) ratios in the presence of tumor growth factor-β (TGF-β) (1 ng/ml)(Bio-Techne, Abingdon, OX, USA) and recombinant interleukin-2 (IL-2) (100 U/ml) (Chiron, Emeryville, CA, USA) in X-Vivo-15 (BioWhittaker, Walkersville, MD, USA) media supplemented with 10% fetal bovine serum (Valley Biomedical) for 72 h.
The iTregs were cultured in the same media with the addition of recombinant IL-2 (300 U/ml) at the concentration of 0.5 × 10⁶ cells/ml. IL-2 (300 U/ml) was added every 2 or 3 days. iTregs were treated with miR inhibitor or miR mimic (Ribobio Corporation, Guangzhou, China) and renewed together with IL-2 on point days. The inhibitor group was treated with miR-142-3p inhibitor (100 nM), while the mimic group was treated with miR-142-3p mimic (50 nM). Cells were collected and tested as described.