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

Forced over-expression of <i>ALK, PHOX2B</i> and <i>PHOX2A</i> in HeLa and NB cell lines.

<p>A) Transcription levels of the <i>ALK</i>, <i>PHOX2B</i> and <i>PHOX2A</i> genes were evaluated in HeLa cells 48 hours post-transfection with the corresponding gene-specific cDNA expressing vectors by real-time RT-qPCR. Besides the dramatic increase of gene transcripts by each respective transfectants, <i>ALK</i> expression results enhanced by the <i>PHOX2</i> genes over-expression. Values are the mean ± s.d. of N = 3 independent experiments performed in duplicate. B) Upper (I) and lower (II) lanes from immunofluorescence analysis report examples of HeLa cells transfected with the PHOX2B-Myc expression construct. From left to right images show DAPI stained cell nuclei (blue), staining for the PHOX2B-Myc protein (green), and staining for the ALK protein (red). The most distal image is the merge of the three nearby figures. C) Western blot evaluating protein amounts of ALK and PHOX2B in HeLa cells at 72 hours post-transfection with gene-specific cDNA constructs. A consistent transcript increment of each gene is observed but ALK starts to be expressed also following the forced expression of PHOX2B-Myc protein. Gel was loaded with 100 µg of total protein extracts except for evaluation of ALK in <i>ALK</i>-transfected cells and PHOX2B in <i>PHOX2B</i>-transfected cells, for which 1∶10 (10 µg) of protein extracts was loaded to avoid over-saturation of autoradiograph films. D) Western blot evaluating protein amounts of ALK in ACN cells (left) at 72 hours post-transfection with the PHOX2B-Myc construct, in a clone of IMR-32 cells stably expressing the same PHOX2B-Myc fusion protein (right). A marked increment in ALK expression is detected following PHOX2B-Myc expression in transient –transfected ACN cells compared to both native and mock-transfected cells and in a clone of IMR-32 cells, stably expressing PHOX2B-Myc, compared to native cells. An anti-cMyc antibody was specifically used to distinguish PHOX2B-Myc fusion protein from endogenous PHOX2B of NB cells (lower blots). E) Two stable IMR-32 clones, one negative (104) and one positive (49) for PHOX2B-Myc expression were analyzed for ALK expression (upper panels); expression of the fusion PHOX2B-Myc protein was assessed by Western Blot using anti –cMyc (middle panels) and the anti-actin antibodies (bottom panels).</p

Gene expression analysis in NB and HeLa cells and correlation analysis.

<p>(A) Relative gene expression analysis of the <i>ALK</i>, <i>PHOX2B</i> and <i>PHOX2A</i> genes, carried out in a panel of NB and in HeLa cell lines by real-time RT-qPCR using a pool of normal tissue RNAs as reference sample (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013108#s4" target="_blank">Methods</a>), shows over-expression of the three genes in all but two NB cell lines tested (GI-ME-N and ACN). (B) X-Y Plots showing a significant correlation between the expression level of <i>PHOX2B</i> and <i>PHOX2A vs.ALK</i> (left) and <i>PHOX2Avs. PHOX2B</i> (right) genes in the analyzed cell lines. Pearson's correlation coefficient indicates a very significant correlation of the three transcription levels <i>vs.</i> each other. Values are the mean ± s.d. of N = 3 independent RT-qPCR analyses performed in triplicate.</p

PHOX2B effect on the <i>ALK</i> promoter sequentially deleted plasmids.

<p>A) Schematic representation of deleted plasmid inserts, progressively shorter from the entire wt <i>ALK</i> promoter region considered (−671 bp), down to the so called deletion 2 (del2; −351 bp), and to the so called deletion 3 (del3; −31 bp) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013108#pone-0013108-g004" target="_blank">figure 4A</a>). The promoter (grey bar), the 5′UTR (black bar) and the ATTA boxes (black circles) are shown. B) Activity of the <i>ALK</i> promoter fragments, expressed as percentage of the activity of the wt construct. Values are the mean ± s.d. of N = 3 independent experiments performed in HeLa cells. C) PHOX2B-mediated induction of the <i>ALK</i> promoter deleted plasmids, expressed as fold increase of the Luciferase activity obtained with respect to the use of the empty vector (pcDNA3.1) on the wt promoter. Values are the mean ± s.d. of N = 3 independent experiments performed in duplicate in HeLa cells.</p

<i>In vitro</i> interaction of PHOX2B with the <i>ALK</i> promoter.

<p>A) EMSAs were performed using probes containing one of the ATTA sites of the region under analysis (ATTA 1, ATTA 2, ATTA 3 and the complex ATTA 4/5). Each labeled probe was incubated in the absence of nuclear extracts (lane 1), with IMR-32 nuclear extracts (lanes 2–4) or the <i>in vitro</i> expressed PHOX2B-Myc fusion protein (lanes 5–7). As negative control the oligonucleotides were also incubated with the <i>in vitro</i> reaction performed using the empty vector pcDNA3.1 M/H (lane 8). The competition experiments were performed in the presence of a molar excess of the unlabeled oligonucleotides (lanes 3 and 6). The anti-PHOX2B or the anti-c-Myc antibodies were added to the samples run in lanes 4 and 7, respectively. On the left, the arrows indicate the specific retarded bands detected; on the right, one or two asterisks indicate the supershifted complexes containing PHOX2B obtained by incubation of IMR-32 nuclear extracts with the anti-PHOX2B antibody (*) or the <i>in vitro</i> expressed protein with the anti-cMyc antibody (**), respectively. The free probes are shown at the bottom of the gels. B) ChIP assay. Chromatin extracted from IMR-32 cells was immunoprecipitated using the antibody against PHOX2B; pre-immune chicken IgY and the anti-acetylated histone H4 antibodies were used as negative and positive controls, respectively. The input represent 0,5% of the total chromatin extract. The precipitated DNA has undergone PCR amplification by using primers bordering the ATTA 3 and the ATTA 4/5 boxes in the <i>ALK</i> promoter.</p

Effect of ATTA 4/5 disruption in competition of PHOX2B binding.

<p>IMR32 nuclear extracts were incubated with the ATTA4/5 probe (lane 2) and competition obtained by adding an excess of: a wild type (wt) probe (lane 3), a probe mutated in both ATTA 4 and ATTA 5 sites (lane 4) or in each of them (lanes 5–6). Incubation without nuclear extracts was regarded as negative control (lane 1).</p

siRNA-mediated silencing of <i>ALK, PHOX2B</i> and <i>PHOX2A</i> in NB cells.

<p>Effects on the transcription level of the <i>ALK</i> (left side graphs), <i>PHOX2B</i> (middle graphs) and <i>PHOX2A</i> (right side graphs) genes after knock-down of the same genes in SHSY-5Y (A), IMR-32 (B) and HTLA-230 (C) cells. Gene-specific knock-down, evaluated 48 hours post-transfection by real-time RT-qPCR analysis, is very effective but also <i>PHOX2</i>-directed siRNAs are able to downregulate <i>ALK</i> at a similar extent (**: <i>P</i><0.01; ***: <i>P</i><0.001). Values are the mean ± s.d. of N = 3 independent experiments performed in duplicate. (D) Gene silencing was confirmed at 72 hours post-transfection by Western blot.</p

Effects of mutagenesis of ATTA 3 and ATTA 4/5 on the PHOX2B-mediated <i>ALK</i> trans-activation.

<p>Left side: schematic representation of the three constructs carrying all the ATTA boxes functional (wt, all four black circles), the ATTA 3 disrupted (ATTA 3 mut, one white circle) or both the ATTA 4 and 5 disrupted (ATTA 4/5 mut, two white circles). Right side: induction of the <i>ALK</i> promoter containing the mutant ATTA 3 and ATTA 4/5 in HeLa cells co-transfected with the PHOX2B expression plasmid are expressed as percentage of the Luciferase activity obtained by cells co-transfected with PHOX2B and the <i>ALK</i> promoter (wt) vectors (wt, arbitrary value = 100). Values are the mean ± s.d. of N = 3 independent experiments (*: <i>P</i><0.05).</p

Sequences of primers employed for real-time RT-qPCR.

<p>Sequences of primers employed for real-time RT-qPCR.</p