PMC

Mitoses are shown by arrows; colored insets indicate eosinophilic cytoplasm, blood vessels, necrosis, nuclear fragmentation, or cytoplasm vacuolization.

Extracts from tumor tissue were analyzed by Western blotting with antibodies against c-MYC, phospho-ERK1/2, ERK1/2, CDC14A, VEGF and β-actin. Representative immunoblots and quantification from three independent experiments (n = 9 mice)of (A) c-MYC, relative to β-actin; (B) phospho-ERK1/2, relative to ERK 1/2; (C) CDC14A and VEGF, relative to β-actin. Data are expressed as means of relative band intensity ± SEM. * p<0.05 versus H1299 (Student's t-test).

(A) Extracts from tumor tissue were analyzed by Western blotting with antibodies against HA, TfR1, ferritin, ferroportin, DMT1 and β-actin. (B) Tumor extracts were analyzed for IRE-binding activity by EMSA with a ³²P-labeled ferritin IRE probe. (C and D) Analysis of TfR1 and H-ferritin mRNA expression by qPCR.

(A) Hierarchical clustering of all differentially expressed genes. Red and green colors represent up- and down-regulation. (B) Venn diagram of all differentially expressed genes. (C) Co-expression network of 178 genes shared by all differential groups. Each gene is represented by a node and a Pearson correlation coefficient above 0.90 between any pair of genes is represented by an edge. Genes are colorized according to their distinct MCL cluster which may reflect a shared biological function.

BALB/c nude mice were injected with parent H1299, HIRP2_wt or HIRP2_Δ73 cells and tumor xenografts were grown for 10 weeks and monitored over time. (A) Representative anesthetized mice before sacrifice; tumor xenografts are shown by arrows. (B–D) Cumulative data from three independent experiments (n = 9 mice for each group) depicting kinetics on tumor xenograft growth (B), mass (C) and volume (D) of isolated tumor xenografts. Data are expressed as mean ± SEM. * p<0.05, ** p<0.01 versus H1299 (Student's t-test).

Parent H1299, HIRP2_wt and HIRP2_Δ73 cells were grown for 3 days in the absence or presence of tetracycline; where indicated, the cells were treated overnight with 100 µM of desferrioxamine (DFO) or hemin. (A) Cytoplasmatic extracts were analyzed by EMSA with a ³²P-labeled ferritin IRE-probe. (B) Analysis of TfR1 mRNA expression by qPCR. (C) Western blotting with antibodies against HA, TfR1 and β-actin. (D) The cells were metabolically labeled with ³⁵S-methionine/cysteine and the synthesis of TfR1 was assessed by quantitative immunoprecipitation. (E) Western blotting with antibodies against HA, ferritin and β-actin. (F) The cells were untreated or pretreated for 4 h with 100 µM hemin and, subsequently, metabolically labeled with ³⁵S-methionine/cysteine; the synthesis of ferritin was assessed by quantitative immunoprecipitation.

A total of 6 BALB/c nude mice were injected with HIRP2_wt cells to form tumor xenografts. Half of the animals were receiving 2 mg/ml tetracycline in the drinking water throughout the experimental period, starting 4 days before injection. (A) Representative anesthetized mice from the two groups before sacrifice (tumors shown by arrows). (B) Kinetics on tumor xenograft growth. (C) Mass and (D) volume of isolated tumor xenografts. (E) Tumor tissue extracts were analyzed by Western blotting with antibodies against HA, to detect expression of the IRP2 transgene, and β-actin, as loading control. Data are expressed as mean ± SEM. * p<0.05, ** p<0.01 versus HIRP2_wt (Student's t-test).