FHC silencing increases cell proliferation.

<p>(A) Western blot analysis for FHC was performed on 50μg of total proteins extracted from FHC-silenced H460 (H460^siFHC) or from H460 control cells (H460^siRNA). Blots are representative of three independent experiments. γ-Tubulin was used as a loading control. The graph represents the mean of the optical densities (*<i>p</i> < 0.05 compared with H460^siRNA). (B) Real-time PCR analysis of FHC mRNA amounts performed on total RNA from H460^siFHC and H460^siRNAcells. Results are representative of three different experiments (*<i>p</i><0,05 compared with H460^siRNA). (C) H460^siRNAand H460^siFHCcells were incubated for 15 min with 20 μM of 2’-7’-DCF and washed with HBSS solution. Fluorescence was measured at 485 nm and 535 nm after60 min. (D) Cell proliferation was assessed using the MTT method as indicated in the Materials and Methods section. Final results represent mean ± SD of three independent experiments each performed in octuplicate (*<i>p</i>< 0.05 compared with H460^siRNA). (E) Western blot analysis for CCND1, p53 and pAKT were performed on 50μg of total proteins extracted from H460^siFHC and H460^siRNA. Blots are representative of three independent experiments. γ-Tubulin and AKT were used as loading controls.(F) Western blot analysis for FHC was performed on 50μg of total proteins extracted from FHC-stably silenced H460 (H460^shFHC) or from H460 control cells (H460^shRNA). Blots are representative of three independent experiments. γ-Tubulin was used as a loading control. The graph represents the mean of the optical densities (*<i>p</i> < 0.05 compared with H460^shRNA). (G) Cell proliferation of stably silenced cells was assessed using the MTT method. Final results represent mean ± SD of three independent experiments each performed in octuplicate (*<i>p</i>< 0.05 compared with H460^shRNA).</p

FHC is required for caffeine modulation of H460 proliferation.

<p>(A) Real-time PCR analysis of FHC mRNA amounts was performed on total RNA from H460^siFHC and H460^siRNA cells treated with the indicated doses of caffeine. Results are representative of two different experiments (*<i>p</i> < 0.05 of each caffeine concentration compared with untreated cells; NS not significant). (B) Cell proliferation was assessed using the MTT method on H460^siFHC and H460^siRNAcells treated with caffeine at the indicated doses. Final results represent mean ± SD of three independent experiments each performed in triplicate (*<i>p</i> < 0.05 of each caffeine concentration compared withuntreated cells; NS not significant). (C) Direct cell counting of H460^siFHC and H460^siRNAcells treated with caffeine at the indicated doses. Final results represent mean ± SD of two independent experiments (*<i>p</i> < 0.05 of each caffeine concentration compared with untreated cells; NS not significant). (D) Western blot analysis forCCND1, p53 and pAKT were performed on 50μg of total proteins extracted from H460^siFHC and H460^siRNA treated with 80μM caffeine or untreated. γ-Tubulin and AKT were used as loading controls.</p

Caffeine reduces H460 cell proliferation.

<p>(A) Cell proliferation was assessed using the MTT method as indicated in the Materials and Methods section. Final results represent mean ± SD of three independent experiments each performed in triplicate (*<i>p</i> < 0.05 of each caffeine concentration compared with NT-untreated cells). (B) Direct cell counting of NT-untreated and 80μM caffeine<b>-</b>treated cells. The results are the mean of two independent experiments (*<i>p</i> < 0.05 compared with NT-untreated cells). (C) and (D)Western blot analysis for CCND1, p53 and pAKT were performed on 50μg of total proteins. Blots are representative of three independent experiments. γ-Tubulin and AKT were used as loading controls. (E) DNA was extracted from cells and analyzed on a 2% agarose gel as described in Materials and Methods. The image is a representative experiment.</p

MRE <i>in silico</i> analysis.

<p>Panel A. The 3’UTRs of FHC and FTH1P3 contain a highly conserved domain (in white) in which several common MRE, indicated by vertical bars, have been identified. The corresponding miRNAs are indicated above the vertical bars. Panel B. Binding of common targeting miRNAs to FHC and FTH1P3 transcripts.</p

Four miRNAs are significantly modulated by FHC amounts.

<p>A) Real-time PCR analysis of FHC mRNA performed on total RNA from K562 shRNA, K562 shFHC and K562^shFHC/pc3FHC. Results are representative of two different experiments. B) TaqMan analysis of hsa-miR-125b, hsa-let-7f, hsa-let-7g, hsa-let-7i in K562 shRNA, K562 shFHC and K562^shFHC/pc3FHC. Results are representative of two different experiments. <b>N.S.: Not Significant</b></p

Four miRNAsare significantly up-regulated after H ferritin silencing.

<p>Four miRNAsare significantly up-regulated after H ferritin silencing.</p

The two highest scoring networks identified by IPA, that correlate genes target of the miRNAs differentially expressed after FHC silencing.

<p>Ingenuity Pathway Analysis was used to investigate the networks potentially affected by the down-regulated genes. (A) Cell Death and Survival, Hematological System Development and Function, Hematopoiesis” is the highest scoring network with a significance score of 37 and 18 focus molecules (B) DNA Replication, Recombination and Repair, Cell Cycle, Cancer” has a significance score of 32 and 16 focus molecules. The target down-regulated genes are shaded in green. Intensity of shading correlates with the degree of down-regulation. A solid line represents a direct interaction between two genes, while a dotted line indicates an indirect interaction.</p

FHC silencing in K562 cells reduces proliferation rate via RAF1/MAPK pathway inhibition and is associated with <i>c-Myc</i>down-regulation.

<p>A) Real-time PCR of <i>RAF1</i> mRNA performed on K562 shRNA, K562 shFHC and K562^shFHC/pc3FHC. Results are representative of two different experiments B) Western Blot analysis for pERK1/2 was performed on 50μg of total protein extract from K562shRNA and K562shFHC cells. Total ERK1/2 was used as loading control. Results are representative of three different experiments. C) Western Blot analysis for pERK1/2 and FHC was performed on 50μg of total protein extract from K562shRNA and K562shRNA+FHC. Total ERK1/2 and γ-Tubulin were used as loading controls. Results are representative of two different experiments. D) Equal number of starved silenced and un-silenced cells were plated into a 96-well plate, incubated for 72 h and analysed by MTT assay. Proliferation of FHC-silenced cells is reduced of about 35% compared to controls. Data are presented as mean ±standard deviation. E) Real-time PCR of c-<i>Myc</i> mRNA performed on K562 shRNA, K562 shFHC and K562^shFHC/pc3FHC. Results are representative of two different experiments.</p

miRNA-mRNA interaction networks.

<p>miRNA-mRNA interaction networks built by Cytoscape. We identified a total of 108 miRNA-mRNA significantly negatively correlated interaction. The four up-regulated miRNAs are colored in red and the 91 down-regulated target mRNAs are in green. let-7i is correlated with 13 transcripts; let-7f and let-7g with 20 and 25 transcripts, respectively; miR-125b negatively correlates with 50 transcripts. The majority of genes are supported targets of only one specific miRNA, whereas 15 genes are putatively regulated by two or more distinct up-regulated miRNAs.</p

Top 9 molecular networks predicted by IPA, by analysis of genes with expression profiles significantly negatively correlated with that of miRNAs differentially expressed after FHC silencing.

<p>Top 9 molecular networks predicted by IPA, by analysis of genes with expression profiles significantly negatively correlated with that of miRNAs differentially expressed after FHC silencing.</p