Ccl 186

IMR-90 cells were purchased from ATCC (CCL-186) at PD25 (~passage 12) and cultured in EMEM supplemented with 10% FBS, penicillin and streptomycin in a humidified atmosphere of 5% CO₂ at 37 °C. The cells were trypsinized and expanded every 3–4 days and their numbers were counted in a hemocytometer to calculate the PDs of each culture. The doubling time (in hours) was calculated with the following formula = h*ln(2)/ln(c2/c1), where c is the number of cells at each time of collection, and ln is a neperian logarithm. SEN fibroblasts were obtained by sub-culturing the same IMR-90 cells until they failed to reach confluency after 2 weeks of culturing (~PD75) and used as positive control for SAFs. Paraquat dichloride hydrate (PQ) induced senescence was triggered as previously described, with minor modifications for human fibroblasts (IMR-90)50 (link). Briefly, early passage HPF were exposed to 30 uM of PQ for 10 days and then processed for RNA isolation and SA-β-gal staining. Bleomycin (Bleo) induced senescence was triggered as previously described31 (link). Briefly, early passage HPF were exposed to 20 μg/ml Bleo for 2 h with a recovery period of 10 days, after which they were processed for RNA isolation and SA-β-gal staining.

Primary human skin fibroblasts (LF1) (ATCC, PCS-201-013), U-2 OS osteosarcoma (ATCC, HTB-96), human embryonic kidney HEK293T (293T) (ATCC, CRL-3216), normal Human Lung Fibroblasts (IMR90) (ATCC, CCL-186), and 3T3-L1 mouse preadipocytes (ATCC, CL-173) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% of fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/streptomycin. D. melanogaster cell line S2 were cultured in Schneider medium (Sigma) supplemented with 10 % FBS. All the cell lines have been continuously tested negative for mycoplasma contamination using DAPI staining. Mammalian cell lines were authenticated from ATCC. After obtaining them, many batches were frozen and proper cell morphology and proliferation rates were continuously checked. None of the cell lines cited above are listed in the database of commonly misidentified cell lines, ICLAC.

HFL-1 (CCL-153; ATCC, Manassas, VA, USA), IMR-90 (CCL-186; ATCC), and WI-38 (CCL-75; ATCC) human lung fibroblasts and A549 human lung adenocarcinoma cells (CCL-185; ATCC) were used in this study. HFL-1 was cultured in F12–K medium (21127022; Thermo Fisher Scientific, Waltham, MA, USA). IMR90 and WI-38 cells were maintained in Eagle's minimum essential medium (10-009-CV; Corning Cellgro, Manassas, VA, USA). A549 cells were cultured in the Roswell Park Memorial Institute 1640 medium (10-040-CVRC; Corning Cellgro). All media were supplemented with 10% fetal bovine serum (16000044; Thermo Fisher Scientific) and 1% antibiotic-antimycotic (15240062; Thermo Fisher Scientific). Cells were grown in 5% CO₂ at 37 °C. VitC (l-ascorbic acid; A92902; Sigma-Aldrich, St. Louis, MO, USA) and NAC (A9165; Sigma-Aldrich) were dissolved in distilled water, vitamin D (VitD) (Cholecalciferol; C9756; Sigma-Aldrich) was dissolved in ethanol, and vitamin E (VitE) (DL-α-Tocopherol acetate; T3376; Sigma-Aldrich) was dissolved in chloroform, according to the manufacturer's instructions.

Isotopically labeled glucoses (1-³H, 2-³H and 6-³H) were purchased from Perkin Elmer and 1-¹⁴C and 6-¹⁴C labeled glucose were purchased from American Radiolabeled Chemicals. H₂¹⁸O isotopically labeled water was purchased from Cambridge Isotope Laboratories. Hexokinase, pyruvate kinase, phosphoglucomutase, UDP-glucose pyrophosphorylase and inorganic pyrophosphatase were purchased from Millipore-Sigma. Isofagomine D-tartrate was purchased from Carbosynth. Deoxynojirimycin, castanopermine, swainsonine and australine hydrochloride were purchased from GlycoFine chemicals. D-gluconon-1,5- lactone was purchased from Frontier Scientific. Uridine disphsophoglucose disodium salt (UDP-glucose) was purchased from Abcam. Uridine 5’diphosphate disodium salt hydrate (UDP) was purchased from Millipore-Sigma. Vero, IMR90, CHO-K1 and HT-29 cells were purchased from ATCC (CCL-81, CCL-186, CCL61 and HTB-38). Full-length TcdA and TcdB toxins were purchased from List Biological Laboratories Inc. FITC-Annexin V staining kit was purchased from Biolegend. All other reagents were of analytical grade and purchased from commercial sources.

Human BJ primary foreskin fibroblasts (ATCC, CRL-2522), human IMR90 fetal lung fibroblasts (ATCC, CCL-186), human HEK 293T embryonic kidney cells (Intercell, AG) and mouse MEFs embryonic fibroblasts Pml^+/+ or Pml^-/- (from Dr. Lallemand-Breitenbach, and whose cell identity was authenticated by STR profiling) were cultivated in DMEM medium (Sigma-Aldrich, D6429) containing 10% of fetal calf serum (FCS) (Sigma-Aldrich, F7524), 1% of penicillin/streptomycin (Sigma-Aldrich, P4458), at 37 °C under 5% CO2 and humid atmosphere. All cell lines were tested negative for mycoplasma contamination. Drugs and molecules used for cell treatments are described in the Key Resources Table in Appendix (duration is mentioned in the main text). BJ, MEFs or IMR90 cells stably expressing transgenes were obtained by retroviral or lentiviral transduction as in Cohen et al., 2018 (link). Transduced cells were then selected 24 h later by adding the appropriate selective drug (puromycin (Invivogen, ant-pr) at 1 μg/mL or blasticidin (Invivogen, ant-bl) at 5 μg/mL).