Ecb2071l

Normal Human Dermal Fibroblasts (NHDF) were purchased from Sigma (C-12302, Sigma). Cells were cultured in EMEM (ECB2071L, Euroclone), 10% FBS (ECS0180L, Euroclone), 1% NEAA (ECB3054D, Euroclone), 1% 200 mM glutamine (ECB3000D, Euroclone), 1% Penicillin/Streptomycin (ECB3001D, Euroclone). Human aortic smooth muscle cells (HSMC) were purchased from the American Tissue Culture Collection (PCS-100-012, ATCC, Manassas, USA). Cells were cultured in ATCC Vascular Cell Basal Medium (PCS-100-030, ATCC; 500 mL added with 500 μL ascorbic acid, 500 μL rh EGF, 500 μL rh insulin and rh FGF-b, 25 mL glutamine), 5% FBS (ATCC Vascular Smooth Muscle Growth Kit), and 5 mL Penicillin-Streptomycin 100X (Euroclone, Milan, Italy). Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (Bend, OR, USA) and routinely grown in Endothelial Growth Medium (EGM^™-2) as indicated by the provider. The neuroblastoma cell line SH-SY5Y was purchased from Sigma (ECACC 94030304, Sigma) and cultured in EMEM (ECB2071L, Euroclone) and HAM’S F-12 (ECB7502L, Euroclone) 1:1, 10% FBS (ECS0180L, Euroclone), 1% NEAA (ECB3054D, Euroclone), 1% 200 mM glutamine (ECB3000D, Euroclone), 1% Penicillin/Streptomycin (ECB3001D, Euroclone). All cultures were maintained at 37°C in a 5% CO₂ incubator.

HeLa cells were cultured in MEM (catalogue no. ECB2071L, Euroclone) supplemented with 10% inactivated fetal bovine serum (FBS) (catalogue no. ECS0180L, Euroclone), 2 mM glutamine (catalogue no. ECB3000D, Euroclone), penicillin (100 IU ml⁻¹) and streptomycin (100 μg ml⁻¹) (catalogue no. ECB3001D, Euroclone) and maintained at 37 °C and 5% CO₂. RagC KO HeLa cells and RagA KO HeLa cells were previously generated and described in ref. ^{7 (link)}. Flp-In 293 T-REx cells (catalogue no. R78007 Thermo Fisher) were grown in DMEM (catalogue no. D6429 Sigma-Aldrich), supplemented with 10% (vol/vol) FBS (catalogue no. 10270 Thermo Fisher), 100 U ml⁻¹ penicillin and 100 µg ml⁻¹ streptomycin (catalogue no. P0781 Sigma-Aldrich), 100 µg ml⁻¹ Zeocin (catalogue no. ant-zn-5b InvivoGen, Toulouse, France) and 15 µg ml⁻¹ Blasticidin (ant-bl-5b InvivoGen). Cell lines were validated by morphological analysis and routinely tested for absence of mycoplasma.

Primary human dermal fibroblasts (HuDe) were purchased from the Istituto Zooprofilattico Sperimentale (Brescia, Italy) as a pooled sample from female subjects (40 years).
Cells were cultured in minimum essential medium with Earl Salts (ECB2071L, Euroclone), with 10% fetal bovine serum, 1% penicillin (10,000 U/mL), 1% streptomycin (10 mg/mL) and 1% Amphotericin B (250 ug/mL) maintained in standard culture conditions at 37 °C in CO₂ under a humidified atmosphere.
The cells were plated at a density of 11,000/cm², the medium was replaced every two days, and cultures were passaged when cell density reached 80% confluence. Experiments were conducted with 8P fibroblasts.
Stress-induced premature senescence (SISP) was induced using DNA damaging agent mitomycin (MMC) 75 nM for one week. Subsequently, proliferative and MMC treated fibroblasts were analyzed by RT-PCR for p16^ink4a and p21^Cip1 as well as senescence phenotype using Senescence Associated β-galactosidase staining.