GoKawiilCell culture and treatments
Human embryonic lung MRC5 fibroblasts (ATCC) and IMR90 fibroblasts (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (Sigma Aldrich, D5796) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U ml−1 penicillin, 100 μg ml−1 streptomycin and 2 mM l-glutamine. The cultures were maintained at 37 °C in an atmosphere of 5% CO 2 . MRC5 fibroblasts were grown under atmospheric oxygen conditions, and IMR90 fibroblasts were cultured under low-oxygen (3%) conditions. Cells were tested regularly for mycoplasma contamination.
For lentiviral transduction HEK293T cells (ATCC) were used, cultured in DMEM without antibiotic and supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 2 mM l-glutamine.
Stress-induced senescence was triggered by exposing cells to 20 Gy of X-ray irradiation and collected between 10 and 12 days post-irradiation. Chemotherapy-induced senescence was performed by treated cells with 250 nM of doxorubicin (MedChemExpress, HY-15142) for 24 h and collected 12 days after treatment. Replicative senescence was performed through serially passaging until the cells reached their replicative limit. Senescence was verified through the presence of p16 and p21, lack of proliferation and the expression of SASP genes.
For chronic acetate treatment, MRC5 or Parkin IMR90 cells were treated with 20 mM of sodium acetate solution (Sigma, S7899) for 10 to 12 days, with media refreshing every 48–72 h.
For chronic citrate treatment, MRC5 were supplemented with 10 mM of sodium citrate (Sigma, W302600) for 10 to 12 days, with media refreshing every 48–72 h.
For SLC25A1 and MPC pharmacological inhibition, MRC5 fibroblasts were irradiated with 20 Gy X-ray irradiation and treated with CTPI2 (Selleckchem, S2968) or UK5099 (Sigma, PZ0160) at the indicated concentrations (15 or 30 μM for CTPI2 and 100 μM for UK5099). CTPI2 and UK5099 were added one day after irradiation and maintained in the cell culture medium for 12 days (refreshed every 48–72 h). The same protocol was used for STING pharmacological inhibition where cells were treated with SN011 (Cayman Chemical, NC2044999) at concentration of 10 μM. For acetyl-CoA measurements, proliferative MRC5 were treated or not with 20 mM of sodium acetate-1-C13 (Sigma, 279293) or sodium acetate C12 (Sigma, S7899) and collected after 3 h. The analysis was performed in 4 million cells per condition.
Parkin-mediated mitochondrial clearance
Parkin-mediated mitochondrial clearance was performed as previously described. In summary, proliferating or irradiated Parkin-overexpressing IMR90 fibroblasts were treated with 12.5 μM CCCP (Sigma Aldrich, C2759) one day post-irradiation (day 1) for a duration of 48 h, with CCCP being replenished every 24 h (day 1, day 2). Acetate was added at day 3 when mitochondria were cleared, and cells were collected at day 12 (media refreshed every 48–72 h).
Lactate dehydrogenase cytotoxicity assay
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