Cell proliferation assay.

<p>Cell counts at three different time points (24, 48 and 72 hours) after transfection of HeLa cells with Ile60Asn-tGFP-fused parafibromin (<b>Ile60Asn</b>), wild-type-tGFP-fused parafibromin (<b>WT-CDC73</b>) or tGFP-vector (<b>tGFP</b>).</p

Western blotting analysis of proteasomal degradation and stability of Ile60Asn parafibromin.

<p><b>A</b>. Western blotting for parafibromin (with anti-<i>CDC73</i> antibody) in HeLa cells in absence (−) or in presence (+) of the proteasome inhibitor MG132. <b>B</b>. Western blotting for parafibromin in HeLa cells after treatment with CHX (0 = no treatment) and harvesting at the indicated time points. <b>C</b>. Graphical representation of the stability of wild-type parafibromin (WT-CDC73) and Ile60Asn mutant at different time points after treatment with CHX.</p

Western blotting for parafibromin and tGFP 48 hours post-transfection.

<p>Immunoblotting for parafibromin in HEK293A (<b>A</b>) and HeLa cells (<b>B</b>) transfected with Ile60Asn-tGFP-fused parafibromin <b>(Ile60Asn</b>), wild-type-tGFP-fused parafibromin (<b>WT-CDC73</b>) or empty vector (<b>tGFP</b>). Nontransfected cells (<b>Neg ctrl</b>) have been used as a negative control. <b>C</b>. Western blotting with an anti-tGFP antibody has been performed as a control on Ile60Asn and wild-type-parafibromin-transfected cells and on negative control cells. ∼61 kDa: band showing proteolytic degradation of the exogenous parafibromin-tGFP, which generated a protein fragment of approximately the same size of endogenous parafibromin. ∼26 kDa: band corresponding to unconjugated tGFP molecular weight.</p

<i>CDC73</i> sequencing and anti-parafibromin immunohistochemistry in the mandibular tumor.

<p><b>A.</b> Electropherograms of <i>CDC73</i> exon 2 PCR amplicons from tumor DNA after subcloning. Upper panel: allele carrying only the somatic c.179 T>A transversion; lower panel: allele carrying the germline c.(136_144)del5 frameshift mutation. The nucleotides in the wild- type sequences involved in the frameshift deletion and in the single nucleotide substitution are underlined with black lines. <b>B</b>, <b>C</b>. Anti-parafibromin immunostaining in the ossifying fibroma of the jaw. Original magnifications: 10X (A) and 63X (B). <b>D</b>, <b>E</b>. Anti-parafibromin immunostaining in positive controls: human normal parathyroid tissue (<b>D</b>) and sporadic parathyroid adenoma with wild-type <i>CDC73</i> (<b>E</b>). Original magnification: 10X.</p

Real-time RT-PCR for <i>CDC73</i> mRNA in transfected and nontransfected HEK293A cells.

<p>Histogram representing the <i>CDC73</i> mRNA fold change in HEK293A cells transfected with Ile60Asn-tGFP-fused parafibromin (<b>Ile60Asn</b>), wild-type-tGFP-fused parafibromin (<b>WT-CDC73</b>) or empty vector (<b>tGFP</b>). Nontransfected cells (<b>Neg ctrl</b>) have been used as a negative control.</p

Western blotting for cyclin D1 and c-myc 48 hours post-transfection in HEK293A cells.

<p>Immunoblotting for cyclin D1 and c-myc in HEK293A cells transfected with Ile60Asn-tGFP-fused parafibromin (<b>Ile60Asn</b>), wild-type-tGFP-fused parafibromin (<b>WT-CDC73</b>) or tGFP-vector (<b>tGFP</b>). Nontransfected HEK293A cells (<b>Neg ctrl</b>) have been used as a negative control.</p

Additional file 3: of EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition

Effects of CHS-828 and chemotherapeutics on protein translation. A) Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of CHS-828. Caspase 3/7 activity was quantified (using 5 μM of Camptothecin for 4 h as a positive control of apoptosis) and relative ATP levels were determined and then normalized to the number of viable cells. The levels of total AMPK, p-AMPK, total EIF2A and p-EIF2A, total 4EBP1, p-4EBP1 were evaluated by WB. Histogram shows the densitometric analysis of p-AMPK and p-EIF2A (* indicates p-value <0.05). Mean and SD of a biological triplicate. B) Jurkat cells were treated with the indicated concentration of drugs for 48 h and cell viability was measured by Cell Titer Glo. Data are represented as mean and SD of three independent experiments. C) Click-it chemistry based on the incorporation of an aminoacid analog (AHA) was used to monitor protein synthesis. Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of FK866, Rapamycin (RAPA), Doxorubicin (DOXO), Cisplatin (CIS) and Dexamethasone (DEXA). The histogram quantifies the % of AHA positive cells (active protein-synthesizing cells) in the viable cell population. Flow-cytometry experiments were carried out on two biological replicates and statistics were based on acquisition of 20000 events/sample. D) Jurkat cells were treated as in C and the level of p-EIF2A and p-4EBP1 was evaluated. Histogram shows the densitometric analysis of p-EIF2A (* indicates p-value <0.05). Mean and SD of a biological triplicate. E) Primary B-CLL cells were treated for 48 h with or without 30 nM FK866 in the presence or absence of 1 mM NA. Histogram shows the densitometric analysis of p-AMPK/AMPK. (PDF 691 kb