Fmk006

Human dermal fibroblasts (fibroblasts established from a skin biopsy) from five control subjects and five patients who carried the HGPS p.Gly608Gly mutation were obtained from the Coriell Cell Repository. Cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 15% fetal bovine serum (Life Technologies), 2 mM l‐glutamine (Life Technologies), and 1× penicillin–streptomycin (Life Technologies) at 37°C in a humidified atmosphere containing 5% CO₂. Testing for mycoplasma contamination was performed regularly. Fibroblasts were cultured in the presence or absence of MG132 (474790, Merck Chemical LTD), MG115 (SCP0005, Sigma), MG262 (I‐120‐200, R&D Systems), bortezomib (S1013, Euromedex), carfilzomib (S2853, Euromedex), chloroquine diphosphate crystalline (C6628, Sigma), bafilomycin A1 (B1793, Sigma), caspase‐6 inhibitor Z‐VEID‐FMK (FMK006, R&D Systems), pan‐caspase inhibitor Z‐VAD‐FMK (FMK001, R&D Systems), leptomycin B (L2913, Sigma), 2‐D08 (SML1052, Sigma), and ginkgolic acid C15:1 (74741, Sigma). The experiments were performed on fibroblasts of HGPS patients and healthy subjects matched for age and passage.

After 21 days, differentiated cells were used for caspase inhibition. The cells were seeded at a density of 5000 cells per cm² and cultured for 6 days (as reported previously^{4 (link)}) in the presence of caspase inhibitors: general caspase inhibitor Z-VAD-FMK and inhibitors for individual apoptotic caspases (Caspase-2,-3,-6,-8,-9). Pharmacological inhibitors (FMK001, FMK003, FMK004, FMK006, FMK007, and FMK008, R&D Systems) were added to the culture medium at a concentration of 100 μM, according to the manufacturer’s instructions and previous studies^{4 (link)}. Controls were generated using DMSO as an inhibitor vehicle at the same concentration. The medium with different treatments was changed every 2 days. The effectiveness of the individual caspase inhibitors was verified with Caspase-Glo assays (Supplement 2), and the constant inhibition effect of the FMK inhibitors in cells was confirmed previously^{38 (link)}