Smooth muscle basal medium 2

HASMCs were cultured in smooth muscle growth medium-2 (Lonza), which contains fetal bovine serum (FBS) and various growth factors, and starved for 24 h before stimulation in smooth muscle basal medium-2 (Lonza) devoid of serum or growth factors. Raw264.7 cells were purchased from American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS. BMDMs were isolated from femurs of wild-type mice and cultured in Mφ medium (RPMI 1640 medium supplemented with 10% FBS, 40 ng ml⁻¹ murine Macrophage Colony-Stimulating Factor (M-CSF), 2 mM L-glutamine, 50 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin). For M1 or M2 polarization, Raw264.7 cells or BMDMs were treated with LPS (50 ng ml⁻¹) or IL-4 (20 ng ml⁻¹) for 24 h, respectively. CM of Raw264.7 cells or BMDMs were collected after another 24 h.

Human aortic smooth muscle cells were purchased from LONZA (Portsmouth, NH, USA) and cultured in smooth muscle basal medium-2 (Lonza, Cat #CC-3181) supplemented with SMC growth medium (Lonza, Cat #CC-4149), as described previously (18 (link)). Prior to stimulation, the cells were starved in 0.5 % FBS-containing medium for 16 h. The cells were treated with 20 μmol/L ox-LDL (Thermo Fisher Scientific, Cat #L34357). Time of the exposure to ox-LDL was 24 h for assessment of senescence in immunoblot (detections of p53 and p21) and in Senescence-associated β-galactosidase (SA-β gal) staining, 12 h for mitochondrial autophagy in immunohistochemistry, and 3 h for other experiments. The following inhibitors were used: 20 μmol/L Mdivi-1 (Sigma, Cat #M0199), Drp1 inhibitor; 10 μmol/L candesartan cilexetil (Candesartan) (Sigma-Aldrich, Cat #SML0245), ARB; 10 μmol/L Dabrafenib (Selleck, Cat #S2807), RAF inhibitor; 2 μmol/L PD184352 (Selleck, Cat #S1020), MEK inhibitor; 2 μmol/L SCH772984 (Selleck, Cat #S7101), ERK inhibitor. For the inhibition experiments, cells were treated with each inhibitor for 1 h following the addition of ox-LDL. Control cells were exposed to the same amount of dimethyl sulfoxide (DMSO). The dose of Candesartan was based on the previous report (19 (link)).