Iron assay kit

TNBC cells were collected after being treated with different concentrations of TAMEWs for 24 h. The relative content of iron was evaluated by an iron assay kit (APPLYGEN, Beijing, China) according to instructions. The absorption was detected at 550 nm. Data were normalized according to the protein content.

mTEC1 cells (1 × 10⁷) were harvested after incubation with/without FAC and L-Cit for 24 h. The cells were washed with cold PBS and then lysed with lysis buffer on a shaker for 2 h. The samples were centrifuged at 12,000 r/min for 10 min to remove insoluble materials. An amount of 4.5% potassium permanganate solution was mixed with the buffer of the Iron Assay Kit (Applygen, Beijing, China) at a ratio of 1:1. The reagents were added in the control tube, standard tube, and sample tube. The samples were incubated at 60 °C for 1 h, then cooled and added to iron ion detection reagents. The samples were incubated at room temperature for 30 min. Absorbance was measured at 550 nm by a microplate reader (ThermoFisher, Waltham, MA, USA).

The cells (2.5 × 10⁵/well) transfected with sh-eIF5A, shRNA-NC, pcDNA, or pcDNA-eIF5A were inoculated into a 6-well plate. After 24 h, the cells were treated for 30 min with RIPA lysate, and the supernatant was collected. Then the Fe²⁺, SOD, and MDA contents were detected through Iron assay kit (Beijing Applygen Technologies, China), SOD detection kit, and MDA detection kit (A001-3-2 and A003-1-2, Nanjing Jincheng Bioengineering Institute), respectively.
The cells transfected with sh-eIF5A, shRNA-NC, pcDNA, or pcDNA-eIF5A were treated with erastin (10 μM) [24 (link)] or Ferrostatin-1 (1 μM) [25 (link)] for 24 h. Then the Fe²⁺ contents were detected through Iron assay kit. Ferrostatin-1 (S7243) and erastin (S7242) were purchased from Selleck (Shanghai, China).