Mir 21 mimic

We seeded 293T cells in 24-well plates, cultured then overnight, and transfected them with the wildtype and mutant TRPM7 promoter-luciferase plasmids (0.1 μg per well of pGV272-wt-TRPM7 or pGV272-mt-TRPM7 plasmids) mediated by Lipofectamine 2000 (Invitrogen, CA, United States). Meanwhile, cells were cotransfected with either 0.4 μg miR-21 mimics or 0.4 μg miR-21 NC (Genepharma, Shanghai, China). Transfection efficiency was standardized by cotransfection with 0.02 μg Renilla luciferase reporter (Genechem, Shanghai, China). Both firefly and Renilla luciferase activities were quantified using a dual-luciferase assay system (Promega, E1910).

The cell transfection was performed as described by Yang et al. [13 (link)]. miR-NC and miR-21 mimics were obtained from Gene Pharma Co. Ltd (China). For transfection, 786-O cells were transfected with 50 pmol/mL miR-21 mimics or miR-NC using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For YAP1 functional assays, a YAP1 expression vector was generated by cloning the open reading frame of the YAP1 gene into the pcDNA3.1 vector, and pcDNA3.1-YAP1 plasmids lacking the 3′ untranslated region (3′-UTR) were cotransfected with miR-21 mimics into cells.

The miR-21 mimics, mimics negative control (mimics control), miR-21 inhibitor, inhibitor negative control (inhibitor control) were purchased from Genepharma Inc (Shanghai, China). Cells were transfected with oligonucleotides by Lipofectamine 2000 (Invitrogen, USA) at a final concentration of 100 nM following the manufacturer’s instructions. At the indicated time for various assays cells were collected.

The blank control group, negative control group (transfected with miR-21 negative
sequences), miR-21 inhibitor group (transfected with miR-21 inhibitors), miR-21 mimic
group (transfected with miR-21 mimics) and PDCD4 siRNA group (transfected with PDCD4
siRNAs) were established. The day before transfection, cells were seeded into 6-well
dishes, and then 2 ml of medium was added to each well. Cell density had to be around
50–60% when transfecting. The medium used was then discarded, and cells washed twice
with Opti-MEM I medium. Opti-MEM (11.5 mL) was added to each well. Opti-MEM I medium
(250 µL) was utilized to dilute 5 µL miR-21 inhibitor, miR-21 mimics (Shanghai
GenePharma Co, Ltd.), corresponding negative controls, and PDCD4 siRNAs (Shanghai
GenePharma Co, Ltd.). Cells were developed for 5 min at room temperature until the
final concentration of 50 nM was reached. Lipofectamine 2000 (Invitrogen) was diluted
and mixed carefully with the above diluted transfections, and cultured 20 min at room
temperature. Then, the above mixture was added into each well containing cells and
medium (500 µL/well) and mixed equally; the dish was incubated in 5%
CO₂-incubator at 37°C, and after 6 h, medium was replaced with a fresh
DMEM (Biowest, France) medium containing 10% FBS. Cells were collected after 48–72 h
of transfection.

Human naive CD4⁺ T (CD4CD45RA) cells were isolated by CD4⁺ T Cell Isolation Kit II (Miltenyi, Bergisch Gladbach, Germany). Cells were pretreated with the PTEN inhibitor bpV (HOpic)(100 μM) for 1 h. To over-express or inhibit miR-21 in hPMSCs, miR-21 inhibitor, inhibitor NC, mimics NC, and miR-21 mimics (GenePharma Co. Ltd., Shanghai, China) were used to treat the hPMSCs. By using the IL-2 (2.5 ng/ml) and anti-CD3/CD28 Dynabeads (1 μg/ml) as mitogenic stimulus, 2 µg/ml hPMSC-Exo were added to CD4⁺ T cells (4×10⁶) and cultured at 37°C in 5% CO₂ for 72 h.
Purified hPMSC-Exo were incubated with PKH26 (Sigma-Aldrich) for 5 min at room temperature. The PKH26 labeled hPMSC-Exo were resuspended in PBS and incubated with CD4⁺ T cells at 37°C for 6 h. After counter-stained nuclei with DAPI, the fluorescence image was captured using a laser scanning confocal microscope (FV3000, Olympus Corporation, Japan).

The mir-21 mimics and inhibitor were purchased from GenePharma (Shanghai, China). The upregulation and downregulation of mir-21 was succeeded by transient mir-21 mimics and inhibitor transfection with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). All steps were completed according to the manufacturer's instructions. Briefly, cells were plated at 5×10⁵ per well in 6-well plates and cultured for 24 h. Then the cells were transfected with the mimics or inhibitors of mir-21 or negative control (NC) RNA at a final concentration of 50 nM, using Lipofectamine 2000 and serum-free Opti-MEM medium (GIBCO, Grand Island, NY, USA). After 6 h, the medium was replaced with DMEM with 10% FBS. qPCR evaluated the transfection efficacy at 24 h and 48 h after transfection.

Primary human glomerular endothelial cells (HGECs, ScienCell Company, Beijing Yuhengfeng Agency, China) were cultured in Endothelial Cell Medium containing 10% fetal bovine serum (Endothelial Cell Medium and serum were from ScienCell Company, Beijing Yuhengfeng Agency, China). The cells were used within six passages. Cells in the high insulin concentration group were treated with medium containing different concentrations of insulin: 7.14 ul, 14.28ul, 35.7ul, 71.4ul of Novolin insulin solution (Novo Nordisk, Copenhagen, Denmark) was added to 2 ml ECM medium separately(The ECM medium did not contain any insulin) to form 5 ng/ml, 10ng/ml, 25ng/ml, 50ng/ml high concentration insulin medium. Cells in the high glucose concentration group were treated with medium containing anhydrous glucose (Solarbio, Beijing, China) at 8.3, 11.1, 16.7, or 33.3 mmol/L. D-Mannitol (Solarbio, Beijing, China) was used as a control for osmolarity. HGECs were transfected with miR-21 mimics or miR-21 inhibitor (GenePharma, Shanghai, China) using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). The transfection was performed under strictly aseptic conditions according to the manufacturer’s instructions. The effectiveness of miR-21 mimics and inhibitor was verified by PCR after transfection (Figures 4A, E).