Figure 1.

Differential expression of proteins of iron metabolism in CCA cells. The CCA4, CCLP1 and HUCCT1 human CCA-derived cell lines were cultured as adherent monolayers (MON) or in 3D sphere conditions (SPH). Panel a. Top left, representative immunoblot analysis. Cell extracts were reacted with antibodies against transferrin receptor (TfR1), ferroportin (FPN), ferritin H subunit (FtH) and vinculin. Cropped blots are displayed. The original full blot images can be found in Supplementary Information. The graphs show densitometric quantification of immunoblot analyses. The values were normalized to vinculin and expressed as a fraction of respective MON cells normalized to 1. Mean values ± SEM (n = 6), *p ≤ 0.05, **p ≤ 0.01 vs control MON for each cell line. Panel b. RNA bandshift analysis of IRP activity. Cytoplasmic extracts were incubated with a ³²P-labeled iron-responsive element (IRE) probe and RNA-protein complexes separated on non-denaturing polyacrylamide gels. On the left a representative autoradiogram is shown. A cropped gel is displayed. The original full gel image can be found in Supplementary Information. The graph on the right shows the densitometric quantification of IRPs bands by direct nuclear counting, as described in Materials and Methods; mean percentages ± SEM of control values (n = 6),, **p ≤ 0.01, ***p< 0.001 vs control MON for each cell line. Panel c. TfR1 and FPN mRNA levels were measured by quantitative RT-PCR. Samples were analyzed in triplicate, normalized to the housekeeping gene 18 S and expressed as percentage of respective MON cells normalized to 1. Mean values ± SEM (n = 6), **p ≤ 0.01 vs control MON for each cell line.