Lysotrakerred

Cells grown on coverslips were fixed in 3.7% paraformaldehyde with phosphate-buffered saline (PBS) for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 15 min, blocked with 10% FBS in PBS for 2 h, and incubated with primary antibodies overnight. Antibodies against human LC3 (#2775) or Lamp1 (SC-20011; Santa Cruz Biotechnology, USA) detected autophagosomes or lysosomes, respectively. In addition, cells were washed and incubated with Alexa Fluor 488-conjugated anti-rabbit, 633-conjugated anti-mouse, 488-conjugated anti-mouse, 405-conjugated anti-mouse, 546-conjugated anti-rabbit, or 546-conjugated anti-mouse secondary antibodies (all from Thermo Fisher Scientific) for 2 h and visualized under a confocal microscope (LSM 510; Carl Zeiss, USA). Alternatively, lysosomes were directly labeled by using LysoTrakerRed (L7528; Thermo Fisher Scientific). Lipofuscins were visualized from cells grown on coverslips through confocal microscopy and autofluorescence at a 505-555 nm wavelength and photographed using a FITC filter after a 493 nm excitation. ImageJ analysis software (NIH, USA) counted puncta in the fluorescence images over 0.5 μm².

Erastin (HY-15763), ferrosatatin-1 (HY-100579), N-acetylcystein (HY–B0215) and MitoSOX Red (HY-D1055) were purchased from MedChem Express (Monmouth Junction, NJ). All-trans retinoic acid (ATRA, R2625), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, M2003) and 4′,6-diamidino-2-phenylindole (DAPI, D9542) were from Merck KGaA (Darmstadt, Germany). Anti-CYTL1 (ab129767) antibody was purchased from Abcam (Cambridge, UK). Anti-CSE (12217-1-AP), anti-ATG5 (10181-2-AP), anti-Keap1 (10503-2-AP), anti-Lamin B1 (12987-1-AP) and anti-p53 (10442-1-AP) antibodies were from Proteintech (Chicago, IL). Anti-HA (3724), anti-LC3B (3868), anti-Myc (2276) and anti-SQSTM1/p62 (8025) antibodies were from Cell Signaling Technology (Beverly, MA). Anti-GAPDH (sc-47724), anti-β-tubulin (sc-5274), anti-β-actin (sc-47778), anti-CBS (sc-133154), anti-Nrf2 (sc-365949), and anti-GPX4 (sc-166570) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Lipofectamine 3000 (L3000), Lipofectamine RNAi MAX (13778), the lysosome-specific dye LysoTraker Red (L7528), and Alexa Fluor 488 goat anti-rabbit IgG (R37116) were purchased from Thermo Fisher Scientific (Waltham, MA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).