N acetyl l cysteine nac

The normal and HGPS human skin fibroblast lines were obtained from Progeria Research Foundation (PRF) (detailed information described in Table S1). Both progeria cell lines carry the classic C1824T mutation. All fibroblast cell lines were cultured in MEM (Life Technologies, Carlsbad, California, United States) supplemented with 15% FBS (Gemini Bio‐Products, West Sacramento, CA, USA) and 2 mm l‐glutamine (Life Technologies) at 37 °C with 5% CO₂. Methylene blue (MB; Acros Organics) was dissolved in PBS and added to the growth medium at a final concentration of 10 or 100 nm. N‐Acetyl‐L‐cysteine (NAC; Acros Organics) was dissolved in PBS and added to the growth medium at a final concentration of 1 mm. Fresh medium was provided twice a week, and the cultures were passaged 1:3 at 95% confluency.

The HGPS and normal human skin fibroblast lines were obtained from the Progeria Research Foundation (PRF) and Coriell Institute (detailed information described in Table S1). The progeria cell line carries the classic C1824T mutation. All fibroblast cell lines were cultured in MEM (Life Sciences) supplemented with 15% FBS (Gemini Bio-Products) and 2 mM L-glutamine (Life Sciences) at 37 °C with 5% CO₂. Methylene blue (MB, Acros Organics), N-Acetyl-L-cysteine (NAC, Acros Organics), and MitoTEMPO (mTEM, Sigma) were dissolved in PBS and added to the growth medium at a final concentration of 100 nM, 100 μM, or 100 nM respectively. MitoQ (Kindly provided by Dr. Michael P. Murphy) was dissolved in DMSO and added to the growth medium at a final concentration of 100 nM (Table S2). Fresh medium was replaced two or three times a week, and the cultures were passaged 1:3 at 95% confluence.

The cell viability was examined using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetra-zolium bromide (MTT) assay according to the previous method [23 (link)]. In brief, 1 × 10⁴ cells per well were plated in 96-well plates. After 24 h, the cells were treated with the desired concentrations of genistein with or without N-Benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk, Calbiochem, San Diego, CA, USA), N-acetyl-L-cysteine (NAC, Invitrogen, Waltham, MA, USA) or LY294002 (Cell Signaling Technology, Inc., Danvers, MA, USA). After 48 h, the medium was changed with fresh medium containing 50 μg/mL MTT solution (Invitrogen). After 2 h, the medium was removed and added 100 μL of DMSO. Absorbance at 540 nm was measured using a microplate reader (Molecular Device Co., Sunnyvale, CA, USA). The morphological changes of cells following genistein treatment were observed and visualized by a phase-contrast microscope (Carl Zeiss, Oberkochen, Germany).

VD3, 25VD, and 1,25VD were purchased from Merck Life Sciences (Milan, Italy) and dissolved in ethanol. Protein kinase C (PKC) inhibitor (GÖ6850) and c-Jun N-terminal kinase (JNK) inhibitor (SP600125) were from Cayman Chemical (Ann Arbor, MI, USA). N-Acetyl-L-Cysteine (NAC), MitoTracker Red CMXRos, and CellROX R Deep Red Reagent were from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). JC-1 was from Adipogen Life Sciences (Liestal, Switzerland). Anti-LC3B antibody was from Proteintech (Rosemont, IL, USA), antitubulin antibody was from Santa Cruz Biotechnology (Dallas, TX, USA), and anti-VDAC and antiphospho-(Ser) PKC substrates antibodies were from Cell Signaling Technology (Danvers, MA, USA). MiR05 was purchased from Oroboros Instruments (Innsbruck, Austria). All other reagents, unless otherwise stated, were from Merck Life Sciences (Milan, Italy).