Smc medium

Human aortic vascular smooth muscle cells (HA-VSMCs, ScienCell, USA) were cultured in SMC medium (ScienCell, USA) supplemented with 2% FBS. Human BMSCs were cultured in MSC medium supplemented with 5% exosome-depleted FBS (ScienCell, USA).
To induce calcification, HA-VSMCs were treated with 2.5 mmol/L Pi for 14 days. To observe the contribution of exosomes and NONHSAT 084969.2 on calcification, HA-VSMCs were treated with 100 μg/ml BMSC-Exos or small interfering RNA NONHSAT 084969.2 (si-NONHSAT 084969.2) for 14 days. Finally, calcification and osteogenic transdifferentiation were evaluated.

Human VSMCs were provided by Sciencell (San Diego, CA, USA), and cultured in SMC medium (Sciencell, CA, USA) at 37 ℃ and 5% CO₂. In the process of cell culture, the aseptic operation principle was strictly followed, and the medium was changed every 2–3 days. When the cell growth and fusion were over 90%, 0.25% trypsin (without EDTA) was used for digestion and passage.
Cell transfection was accomplished to control miR-361-5p levels in VSMCs, and sequences of miR-361-5p mimic, the negative control od miR-361-5p mimic (mimic NC), miR-361-5p inhibitor, and its negative control (inhibitor NC) were synthesized by Ruibo biological technology co. (Guangzhou, China). Cell transfection was detailed described in Table 1. The small interfering RNA of TIMP4 (si-TIMP4) was used for the downregulation of TIMP4, which was also synthesized by Ruibo biological technology co. (Guangzhou, China). Lipofectamine 2000 was applied to the cell transfection in line with the specification. 48 h after transfection, cells from each group were collected for follow-up experiments.
Table 1

Conditions of cell grouping

Groups	Cell transfection
Mock	Normal culture
MiR-361-5p mimic	50 nM miR-361-5p mimic
Mimic-NC	50 nM mimic-NC
MiR-361-5p inhibitor	100 nM miR-361-5p inhibitor
Inhibitor NC	100 nM inhibitor NC

RASMCs were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in SMC medium (ScienCell, Cat#: 1101), containing 5% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 g/ml streptomycin. RASMCs within passage 5 to 12 were used for all experiments. RASMCs were stimulated with 100 nM Ang II (Sigma, Cat#: A9525-50MG) for 24 h before harvest. To knockdown PDE4D, PDE4D siRNA (Ribobio, siB180730051733) and control siRNA (Ribobio, siN0000001-1-5) were purchased from Ribobio (Guangzhou RiboBio Co., Ltd., Guangzhou, China). RASMCs were transfected with 200 nM PDE4D siRNA in 5 μl of Oligofectamine (Invitrogen, Carlsbad, CA, USA, Cat#: 12252011) for 48 h. The siRNA transfection efficiency was determined by real-time polymerase chain reaction (RT-PCR), western blot and immunofluorescence assay (Supplementary Fig. 5a–e).

Human aortic smooth muscle cells (HASMCs), purchased from ScienCell (U.S.A.), were cultured in SMC medium (ScienCell, U.S.A.) with 5% CO₂ at 37°C. Passages 4–7 cells were seeded (10⁵ cells/well) onto six-well Flexcell plates coated with collagen I, and when they reached 90% confluence, serum-free medium was added to induce quiescence for 24 h and replaced with fresh complete medium, then physiological stretch (10% elongation, 1 Hz) was applied by use of a computer-controlled Flexcell 5000-Tension apparatus (Flexercell Strain Unit, FlexCell International). The static control cells subjected to the same conditions as the experimental cells, except that they were not exposed to stretch. For inhibition of stretch-induced activation of transcription factors and signaling pathways, pharmacological inhibitors were added to the culture media 1 h before stretch treatment.

Primary HASMCs were obtained from ScienCell Research Laboratories (San Diego, CA, USA). The cells were cultured as monolayers in smooth muscle cell (SMC) medium (ScienCell) containing essential and non-essential amino acids, vitamins, organic and inorganic compounds, hormones, growth factors, trace minerals and 2% fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. For subcultures, the cells were detached using 0.125% trypsin containing 0.01 M ethylenediaminetetraacetic acid (EDTA). The cells used in the present study were from the early passages (passages 2–6). THP-1 cells were from the American Type Culture Collection (ATCC; Manassas, VA, USA) and weres used for the cell adhesion assay with the HASMCs. These cells were cultured in RPMI-1640, and supplemented with 2 mM L-glutamine, 100 mg/ml streptomycin, 100 IU/ml penicillin and 10% FBS.