Smc growth medium

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)).

Primary tissue derived cultures of human LAM cell lines were established in the Department of Medicine, University of Pennsylvania, Pennsylvania, USA (22 (link)). Briefly, LAM cells were dissociated from LAM nodules of transplant patients. Each LAM nodule was used to establish individual cell lines (characterized by alpha smooth muscle actin (α-SMA) expression, mTORC1 activity, HMB45 immunoreactivity, DNA synthesis, and cell migration) (23 (link)). In the current study, four patient-derived individual LAM cell lines were used including LAM-100, LAM-111C, LAM-D9065 and LAM-HUP. As controls, primary cultures of normal, human bronchial smooth muscle cells (BSMC) and normal human lung fibroblasts (NHLF), were purchased from Lonza (Basel, Switzerland). Normal BSMC-s and LAM cell lines were cultured at 37°C, 5% CO₂ in SMC Growth Medium (insulin, hFGF, GA, FBS and hEGF) (Lonza, Basel, Switzerland). Two angiomyolipoma cell lines were also used in the study and cultured at the above-mentioned conditions. The 621-102 (S102)(TSC2-/-) cell line was generated by introduction of E6/E7 (pLXSN 16E6E7-neo) and human telomerase (pLXSN hTERT-hyg) into a primary culture of TSC2 null human angiomyolipoma cells (24 (link)–26 ). The 621-103 (S103)(TSC2+/+) was generated by stable transfection of TRI102 with wild-type TSC2 (pcDNA3.1 TSC2-zeo) into 621-101 cells (24 (link)).