Fig. 2.

Tumor associated macrophages protect HT1080 cells from ferroptosis

(A) A co-culture system with tumor associated macrophages (TAM) was established. PBMCs were isolated and differentiated for 96 h to macrophages. Afterwards they were incubated with UV irradiated MCF-7 cells for additional 96 h and subsequentially co-cultured with HT1080 cells in a transwell set up and then treated with 1 μM RSL3. (B) HT1080 cells were treated with 1 μM RSL3 and 1 μM liproxstatin-1 (Lip) for 4 h and vitality was measured. (C) HT1080 cells alone or in co-culture with TAMs or naïve macrophages (MΦ) were incubated for 24 h, treated with 1 μM RSL3 and 1 μM Lip for 4 h, and vitality was analyzed. Data were normalized to MΦ control. (D) HT1080 cells were co-cultured with TAMs, incubated for indicated time points, and then treated with 1 μM RSL3 for 4 h. Vitality was analyzed and values were normalized to the corresponding DMSO control. (E) TAMs were generated and transfected with siRNA against ceruloplasmin (siCP) or a non-targeting control (NTC). NTC transfected MΦ served as controls. After 48 h, transfected TAMs and MΦ were co-cultured with HT1080 cells for 24 h, which then were treated with DMSO or RSL3 for 4 h. Vitality was measured and data were normalized to NTC TAMs. All data are expressed as mean values ± SEM, *p ≤ 0.05.