Binding of IFI16 protein to supercoiled DNAs.

<p>(A) 100 ng sc pBluescript (lane 1–6) and sc pCMYC (lane 7–12) were incubated with increasing concentrations of IFI16 (molar ratio DNA:protein 1:0 / 1:1.25 / 1:2.5 / 1:5/ 1:10 / 1:20) in binding buffer (5 mM Tris-HCl, pH 7.0; 1 mM EDTA, 50 mM KCl and 0.01% Triton X-100) on ice for 15 min. The electrophoresis ran for 3 h at 100 V at 4°C. (B) 100 ng linear pBluescript (lane 1–6) and linear pCMYC (lane 7–12) were incubated with increasing concentrations of IFI16 (molar ratio DNA: protein 1:0 / 1:1.25 / 1:2.5 / 1:5/ 1:10 / 1:20) in binding buffer (5 mM Tris-HCl, pH 7.0; 1 mM EDTA, 50 mM KCl and 0.01% Triton X-100) on ice for 15 min. The electrophoresis ran for 3 h at 100 V at 4°C.</p

The experimental setup:

<p>1. Total RNA was extracted. 2. Reverse transcription was performed in two different setups i) using random hexamers to collect all possible types of RNA transcripts and ii) using oligo dT primers to gain polyadenylated types of RNA transcripts. 3) Real-time PCR with primers designed to specifically recognize N- and C-terminus of β-galactosidase cDNA was used to amplify sequences at both 5′-end (PCR-1) and 3′-end (PCR-2) of β-galactosidase RNA transcripts.</p

DNA sequences of oligonucleotides.

<p>DNA sequences of oligonucleotides.</p

H/D exchange of IFI16 in response to DNA interaction.

<p>(A) H/D exchange of IFI16 in response to DNA interaction was analyzed in four reactions: IFI16 protein without DNA as a control, IFI16 with single stranded DNA oligonucleotide (SS DNA), IFI16 with double stranded DNA oligonucleotide (DS NHE III) and IFI16 with DNA forming quadruplex structure (Q NHEIII). H/D exchange was quenched at 900 s after addition of deuterium. The graph shows percentage deuteration of individual amino acids of IFI16 calculated as weighted average of corresponding peptides [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157156#pone.0157156.ref053" target="_blank">53</a>]. Shaded area of the graph shows the areas not covered by peptides. The deuteration spectrum is aligned with the domain structure of IFI16 and with prediction of disordered regions (FoldIndex [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157156#pone.0157156.ref054" target="_blank">54</a>]). (B) Structure of the first HIN-A domain (PDB 2OQ0) corresponding to amino acids 198–389 of IFI16 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157156#pone.0157156.ref014" target="_blank">14</a>]. (C) Complex of the second HIN-B domain with DNA (PDB 3RNU) corresponding to amino acids 516–710 of IFI16 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157156#pone.0157156.ref023" target="_blank">23</a>]. In (B) and (C) the helical linker peptide exhibiting the most significant changes in percentage of deuteration in the presence of quadruplex DNA is highlighted in red.</p

CD spectroscopy of quadruplexes and their stabilization by IFI16.

<p>(A) CD spectra of oligonucleotide HTEL in TE buffer after denaturation (blue line), in TE buffer + 50 mM NaCl (red line) and in TE buffer + 50 mM KCl (green line). (B) CD spectra of oligonucleotide NHEIII in TE buffer after denaturation (blue line), in TE buffer + 50 mM NaCl (red line) and in TE buffer + 50 mM KCl (green line). The schematic drawings represent quadruplex structures of HTEL and NHEIII sequences. (C) The effect of recombinant IFI16 on HTEL quadruplex formation in potassium ions. CD spectra description: HTEL oligonucleotide in TE buffer (blue line), HTEL in TE buffer with 50 mM KCl (red line), HTEL in TE buffer + protein buffer with final concentration 3.4 mM KCl (green line), HTEL in TE buffer + IFI16 in protein buffer at molar ratio 1:1 and final concentration 3.4 mM KCl (violet line), IFI16 protein in protein buffer with final concentration 3.4 mM KCl in TE buffer (black line). (D) The effect of recombinant IFI16 on NHEIII quadruplex formation in potassium ions. The same description of curves as in C (NHEIII instead of HTEL). (E) The effect of recombinant IFI16 on HTEL quadruplex formation in sodium ions. CD spectra description: HTEL oligonucleotide in TE buffer (blue line), HTEL in TE buffer with 50 mM NaCl (red line), HTEL in TE buffer + protein buffer with final concentration 3.2 mM NaCl (green line), HTEL in TE buffer + IFI16 in protein buffer at molar ratio 1:2 and final concentration 3.2 mM NaCl (violet line), IFI16 protein in protein buffer with final concentration 3.2 mM NaCl in TE buffer (black line). (F) The effect of recombinant IFI16 on NHEIII quadruplex formation in sodium ions. The same description of curves as in E (NHEIII instead of HTEL).</p

Comparison of IFI16 DNA binding to structurally different DNA targets.

<p>EMSA was performed with 5 pmol of labeled oligonucleotides forming DS from human telomere sequence–DS HTEL (A) and G-quadruplex from one strand of the same sequence–Q HTEL (B), DS from NHE III region from <i>MYC</i> promoter–DS NHEIII (C) and G-quadruplex from one strand of the same sequence–Q NHEIII (D), SS (E) and cruciform (F) and increasing IFI16 concentrations (0 / 1.25 / 2.5 / 5 / 10 / 20 / 40 pmol), incubated in binding buffer (5 mM Tris-HCl, pH 7.0, 1 mM EDTA, 50 mM KCl and 0.01% Triton X-100) at 4°C for 15 min. Samples were electrophoresed on 4% non-denaturing polyacrylamide gel at 50V and 4°C for 3h. (G) Graphical representation of results obtained from densitometry analysis of free DNA bands from gels of IFI16 binding with SS A50, DS NHEIII, Q NHEIII and cruciform DNA targets from three independent experiments with SD. Schemes of DNA structures in A-F are not to scale.</p

Inhibition of expression from extrachromosomal HIV promoter by PCI.

<p>A β-galactosidase reporter assay was used to measure expression from the promoter within the pHIV-lacZ plasmid following transfection into a Tat-expressing cell line (H1299-Tat). Results were compared with expression from Tat-expressing cell line with pHIV-lacZ stably integrated into the cellular genome (H1299-HIV). The requirement for Tat to promote expression from HIV promoter was illustrated in H1299 (Tat-negative) control cells transiently transfected with pHIV-lacZ. The Y axis represents relative β-galactosidase activity and the error bars illustrate the standard deviation of three independent biological replicates.</p

The effect of OCII and Rosc on phosphorylation of RNA polymerase II CTD.

<p>(A) Phosphorylation of RNA polymerase II CTD in wild type p53 cells either with stably integrated SV40 (COS-1) or SV40-negative cells (CV-1) was determined. The phosphorylation of both Ser 2 and Ser 5 was inhibited after 12 h treatment either with 8 µM OCII or 20 µM Rosc in all tested cell lines. (B) Comparison of RNA polymerase II CTD phosphorylation in cells expressing Tat with pHIV-lacZ stably integrated into cellular genome (H1299-HIV) or with pHIV-lacZ as a part of extrachromosomal DNA (H1299-Tat). The phosphorylation of Ser 2 and Ser 5 in both cell lines was inhibited after PCI treatment for 12 h. The numbers represent the fold changes of samples to untreated controls.</p

OCII and Rosc suppress T-antigen expression.

<p>(A) OCII and Rosc suppress T-antigen expression and restore p53–mediated transactivation of p21^WAF−1 during SV40 infection. MCF-7 (wild type p53) cell line infected with SV40 was treated either with 8 µM OCII or 20 µM Rosc, either immediately or 16 h post-infection. (OCII 16 h, Rosc 16 h). Infected cells were harvested at 45 h and then analyzed for the expression of large T-antigen, p53, p21^WAF−1 and actin by immunoblotting. (B) Inhibition of expression from viral promoter integrated into cellular genome. Cell lines CV-1 and COS-1 were treated either with 8 µM OCII or 20 µM Rosc and analyzed for the expression of large T-antigen, p53, p21^WAF−1 and actin by immunoblotting. CV-1 cell line was used as SV40-negative control cell line (no large T-antigen expression). The numbers represent the fold changes of samples to untreated controls.</p

Olomoucine II differs from roscovitine only by an additional hydroxyl group on 6-benzyl group.

<p>Olomoucine II differs from roscovitine only by an additional hydroxyl group on 6-benzyl group.</p