Fitc or cy3 conjugated secondary antibodies

AP staining was performed using the Alkaline Phosphatase Detection Kit (Sigma) according to the manufacturer’s instructions. For immunofluorescence assay, human iPSCs were adhered on slide chambers (Nunc) containing MEFs, fixed with 4% paraformaldehyde (Sigma) for 30 minutes at RT and permeabilized for 10 minutes with 1% TritonX-100 (Sigma). Cells were blocked with 5% BSA for 30 minutes at RT and incubated for one hour at RT or o/n at 4°C with NANOG antibody (Abcam), SOX2 (R&D), SSEA-1 (Chemicon), SSEA-4 (Chemicon), TRA1-60 (Chemicon) and TRA1-81 (Chemicon) diluted in PBS/TBS with 1% BSA. FITC- or Cy3-conjugated secondary antibodies (Sigma) diluted in the same solution was incubated for 1–1.5 hours at RT. Nuclei were counterstained with 1:4 dilution of DAPI mounting medium (Vector Labs). Samples were visualized under an inverted fluorescence microscope.

SHK was purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA). ZVAD-FMK, Necrostatin-1, 5-Fluorouracil and Oxaliplatin were from MedChemExpress (NJ, USA). ZDEVD-FMK and ZLEHD-FMK were from Adooq bioscience (CA, USA). N-acetyl-L-cysteine and L-glutathione were from Beyotime Institute (Shanghai, China). Antibodies were as follows: PARP, Caspase 3, Caspase 8, Caspase 9, RIPK1, Cytochrome C and VDAC1 were from Cell Signaling Technology (MA, USA); AIF, Endonuclease G, GAPDH and PCNA were from Abcam (Cambridge, UK); the goat IgG-HRP secondary antibodies against rabbit and mouse were from Zhongshan Golden Bridge Biotechnology (Peking, China) and FITC or Cy3 conjugated secondary antibodies were from Sigma (MO, USA).