Hoechst 33342

A total of 2 × 10⁵ cells/well MCF-7 cells were seeded in 6-well plates for 24 h. Then, the samples (free DOX, DOX@Fe-PDA/FA-PEG) were added to each well (equivalent DOX concentration of 10 μg/mL) and the cells were incubated at 37°C for an appropriate time at an additional of 24 h. Afterward, the cells were washed with PBS and stained by Hoechst 33342 (Sangon Biotech, Shanghai, China). Confocal laser scanning microscopy (CLSM) imaging was performed on LSM 410 fluorescence microscope (Zeiss, Jena, Germany). The fluorescence signal of DOX was excited at 488 nm and measured at 610 nm. The fluorescence signal stained by Hoechst 33342 was excited at 405 nm and detected at 490 nm.

First, the Karpas 299 (K299) cells were obtained from CoBioer Biosciences Co., Ltd. (Nanjing, China) and cultured in RPMI 1640 medium with 10% (v/v) FBS and 1% penicillin-streptomycin at 37 ^oC in a humid atmosphere with 5% CO₂. The cells density was determined using a hemocytometer before each experiment. Then, K299 cells were seeded on the 48-well culture plate (3 × 10⁴ cells per well) and incubated for 12 h. The transfection was performed according to the Xfect^TM RNA Transfection Reagent instructions (TaKaRa Biomedical Technology Co., Ltd., China). After transfection for 8 h, the cell nucleus was stained with 10 μL of Hoechst 33342 (Shanghai Sangon Biotech, China) at 37 °C for 30 min. The cells were washed twice with 500 µL of 1× PBS by centrifugation at 1000 rpm for 3 min, followed by resuspending with 300 μL of 1× PBS. Finally, the images were recorded and analyzed by Nikon Confocal Microscope A1 (Nikon, Japan).

P53 and α-tubulin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). ACSL4, SLC7A11, and NOX2 antibodies were purchased from Abclonal (Wuhan, China). Horseradish peroxidase (HRP)-conjugated IgG (H + L) secondary antibodies and CellROX Green Reagent were purchased from Thermo Scientific (Waltham, MA, USA). The Gpx4 antibody was obtained from Abcam (Cambridge, MA, USA). The Cytotox 96 nonradioactive cytotoxicity assay kit was purchased from Promega (Fitchburg, USA). Erastin was purchased from MCE (New York, NY, USA). The Lipid Peroxidation (MDA) Assay Kit, and GSSG/GSH quantification kits were purchased from Beyotime (Shanghai, China). Hoechst 33342 and one-step RT-qPCR kits were purchased from Sangon Biotech (Shanghai, China). Ferrostatin-1 (Fer-1) was purchased from Solarbio Science & Technology (Beijing, China). Precast Gel was purchased from Tsingke Biotechnology (Beijing, China). ChamQ Universal SYBR qPCR master mix was purchased from Vazyme Biotech (Nanjing, China). FerroOrange was purchased from Dojindo Molecular Technology (Kyushu, Japan). Gentamicin and other chemicals were from Sangon Biotech (Shanghai, China). All drug concentrations are expressed as the final molar concentration in working buffer.

DOX hydrochloride was purchased from Nanjing Oddfoni Biological Technology Co., Ltd (Nanjing, China). The siRNA^IGF1R with the sequence of 5′-CAUACUGCGCUCUAUAGAUTT-3′ was selected for targeting the IGF1R gene. The double-stranded siRNA^IGF1R and FAM-labeled siRNA^IGF1R (FAM: carboxyfluorescein) were designed and chemically synthesized by GenePharm Co., Ltd (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation and cytotoxicity assay kit, Annexin V-FITC apoptosis detection kit, Hoechst 33342, 4% paraformaldehyde, folate, biotin, RPMI-1640 medium, phosphate-buffered saline (PBS), fetal bovine serum (FBS), bicinchoninic acid (BCA) protein assay kit and polyvinylidene difluoride (PVDF) membrane were provided by Sangon Biotech Co., Ltd (Shanghai, China). Mouse monoclonal IGF1R beta chain antibody and HRP-conjugated beta-actin antibody, HRP-conjugated goat anti-mouse IgG (H + L) were from Proteintech Group, Inc (USA). All other reagents used were analytical grade and ultrapure water (18.25 MΩ) was used throughout the study.