Ab195046

IHC staining of paraffin-embedded tissues with antibodies against FDFT1 (Abcam, ab195046) was performed and scored according to standard procedures. The staining score was determined by two independent pathologists at our center.

Cells seeded on 12-mm glass coverslips (Nunc) were washed twice with PBS and fixed with 4% paraformaldehyde (PFA) for 20 min. PFA was removed by rinsing twice with PBS and cells were permeabilised with 0.1% (v/v) Triton X-100 in PBS (5 min), washed twice with PBS, and incubated in blocking buffer [0.2% (w/v) fish skin gelatin (FSG) plus PBS, 60 min]. Primary antibody [anti-SQS rabbit mAb, ab195046 (Abcam), 1:200] in blocking buffer was added (60 min), the coverslips were rinsed twice with PBS and incubated in the dark (60 min) with Alexa Fluor 488-conjugated secondary antibody (1:400 in blocking buffer). DNA was stained using 5 µg/ml DAPI before mounting in Fluoromount G (Southern Biotech, Birmingham, AL). For Filipin III staining, after fixation with 4% PFA, cells were rinsed three times with PBS before quenching residual PFA with 1.5 mg/ml glycine plus PBS (10 min). Cells were incubated with Filipin III (Santa Cruz Biotechnology, 25 µg/ml in PBS, 30 min) and rinsed three times with PBS. Coverslips were sealed with transparent nail polish. Antibody-bound proteins were visualised at an excitation wavelength of 488 nm and DAPI and Filipin III were imaged at 405 nm using an LSM 710 (Zeiss, Oberkochen, Germany) confocal microscope. Images were processed using ImageJ and Adobe Illustrator software.

We combined the gene expression data of control samples (17 samples) with those of CRC samples (533samples) in the GSE39582 group and performed the Wilcoxon rank-sum test to further compare the differential expression of the 10 prognostic ferroptosis-related genes between the normal and tumor colon tissues. Besides, a total of 75-paired normal/tumor CRC specimens were recruited from Ruijin Hospital (Shanghai, China) following the guidelines set by the Ethical Committee of Ruijin Hospital. The tumor and adjacent normal colon tissues were fixed by 10% formalin and embedded by paraffin. The optimum sections of tissue specimens were selected and deparaffinized and immunohistochemistry (IHC) was implemented as the following antibodies: HAMP (Abcam, ab30760), FDFT1 (Abcam, ab195046), GDF15 (Abcam, ab206414), TFAP2C (Abcam, ab218107).