Fig. 5.

Ceruloplasmin is transferred from TAMs to HT1080 cells

(A) HT1080 cells were incubated with naïve macrophages (MΦ) and tumor associated macrophages (TAM) in a transwell system and mRNA was isolated from HT1080 cells. Ceruloplasmin (CP) mRNA was measured and normalized to TATA box binding protein (TBP). (B) TAMs were transfected with siRNA against CP (siCP) or a non-targeting control (NTC) and co-cultured with HT1080 cells. mRNA was isolated from HT1080 cells and analyzed for CP, which was normalized to TBP. (C) MΦ and TAMs as well as HT1080 cells, either alone or co-cultured with MΦ respectively TAMs, were analyzed for CP protein expression by Western analyzes. (D) Blots from (C) were quantified and data were normalized to lane normalization factor (LNF). CP expression in HT1080 cells alone could not be quantified (n.q.). (E) TAMs were generated and stained with Cell Tracker CMFDA dye. Cells were measured by FACS-imaging. Mean fluorescence intensity (MFI) was compared between unstained and stained TAMs. Each dot in the graph represents the mean of 100.000 single cells. Pictures of three representative cells are shown. Numbers included in the picture indicate the event count of a flow cytometer. (F) HT1080 cells were co-cultured with unstained and Cell Tracker CMFDA stained TAMs for 24 h and measured by FACS imaging. Mean fluorescence intensity (MFI) was compared between HT1080 cells co-cultured with unstained and stained TAMs. The graph was composed as described for (E). (G) Extracellular vesicles (EV) were isolated from MΦ and TAM supernatants and analyzed for CP mRNA content. CP mRNA was normalized to 18s RNA (18s). (H and I) Iron was measured in HT1080 cells, which were co-cultured with MΦ or TAMs and in HT1080 cells alone. (H) shows Fe(II) and (I) total iron. (J) HT1080 cells were co-cultured with MΦ and TAMs and treated with RSL3 for 4 h. Afterwards, HT1080 cells were stained with BODIPY C11 and analyzed by FACS imaging. Pictures of two representative cells are shown. The numbers indicate the event count of a flow cytometer. (K) MFI was compared between RSL3 treated HT1080 cells after co-culture with MΦ and TAMs. The graph was composed as described for (E). All data are expressed as mean values ± SEM, *p ≤ 0.05.