Phosphorimager screen

Primer extension analyses were performed as described (48 (link)) using ³²P-labeled primers listed in Supplementary Table S1. Sequencing reactions using the same primers were performed with the sequencing kit from (Thermo sequenase 785001KT Affymetrix). The products of primer extension and sequencing reactions were resolved on denaturing (urea), taurine polyacrylamide gels according to the manufacturer's instructions. Dried gels were exposed to Phosphorimager screens (Fujifilm) and radioactive cDNAs were detected using a FLA-9500 Phosphorimager (Fujifilm). Obtained data were analyzed and further processed in MultiGauge v.3.0.

After hybridization, Southern blots were exposed to phosphorimager screens (Fuji) and scanned with an FLA7000 scanner (Fuji). The band intensities were quantified using AIDA image analyzer (version 4.50, Raytest). Intensity of each band was calculated as a percentage of total pixel intensity of the lane. At least three biological replicates were performed for each experiment, means and standard deviations are presented in the figures and figure legends.

After hybridization, Southern blots were exposed to phosphorimager screens (Fuji) and scanned with an FLA7000 scanner (Fuji). Scans shown in figures are representative of at least three independent experiments.

PsrA-DNA binding reactions (8 μl) contained 30 mM Tris-HCl, pH 7.6, 1 mM DTT, 0.25 mM EDTA, 125 mM NaCl, 0.5 mg/ml BSA, 10 mM MgCl₂, 1 mM CaCl₂, 1.25% glycerol, 4 nM radiolabeled IR1.2 DNA or 6 nM radiolabeled IR1.1 DNA, and different concentrations of PsrA. Reactions were incubated at room temperature for 20 min. Then, protein-DNA complexes were treated with DNase I (0.01 units) for 5 min at the same temperature. DNase I digestion was stopped by adding 1 μl of 250 mM EDTA. Four microlitres of loading buffer (80% formamide, 1 mM EDTA, 10 mM NaOH, 0.1% bromophenol blue, and 0.1% xylene cyanol) was added to the reaction mixtures. Samples were heated at 95°C for 5 min, and immediately chilled on ice before loading onto 8 M urea-6% polyacrylamide gels. After running, gels were dried and exposed to a Phosphorimager screen (Fujifilm, Japan). The radioactive intensity was visualized by a Fujifilm Image Analyser FLA-3000 and was quantified using the Quantity One software (Bio-Rad) (Solano-Collado et al., 2013 (link); Ruiz-Cruz et al., 2018 (link)).

EMSA were performed according to the previously published protocol (44 ). A HeLa cell nuclear extract was prepared using the NucBuster™ Protein Extraction Kit (Novagen). About 10⁶ cells per sample were collected and resuspended in 75 µl NucBuster Reagent 1 buffer to lyse the cytoplasmic membrane. The nuclear pellet was resuspended in 75 µl NucBuster Extraction Reagent 2, containing 1 μl 100x Protease Inhibitor Cocktail, and 1µl of 100 mM DTT. After centrifugation the supernatant was collected and labeled as nuclear extract and stored at -80°C. The oligonucleotide sequences containing the transcription factor binding sites assayed in this study are listed in Table S1. The double-stranded oligonucleotides were 5’ end labelled using γ- (32 (link))P-ATP and purified as previously described (45 ). The nuclear extract (4 µl) was incubated with the (32 (link)) P-labelled probe (3 µl, 0.05 pmol) in 5 µl of 4x binding buffer, 1 µl Poly(dI-dC), and 7 µl water for 30 min. As a cold competitor 1 µl containing a 10-fold excess of the unlabeled double-stranded oligonucleotide was added to the binding buffer. All samples were loaded and resolved on a 5% native polyacrylamide gel. The result was visualized by exposure of membranes to a PhosphorImager screen (Fuji) followed by signal analysis using the Image One software (BioRad).

RNA was loaded onto a denaturing 1% agarose gel and blotted to a Hybond NX membrane (Amersham Biosciences), cross-linked at 80°C for 1 h, and hybridized as previously described in detail (49 (link)). Hybridization probes were generated by 5′-end labeling with γ- (32 (link)) P-ATP DNA oligonucleotides complementary to the 5’ end of either the coding sequence of the IL-6 mRNA, or the coding sequence of IκBα. Signals were detected by exposure of membranes to a PhosphorImager screen (Fuji) followed by signal analysis using the Image One software (BioRad). The sequence of the oligonucleotides used for hybridization are listed in Table S1.

The lysed plugs were loaded onto 1% agarose gel made in 0.5x Tris-Borate-EDTA (TBE) buffer and electrophoresed at 6 V/cm for 20–24 hours using initial and final switch times of 60 and 120 seconds, respectively, in Bio-Rad CHEF-DRII PFGE system. The gel was dried under vacuum, exposed to a PhosphorImager screen and scanned in FLA-3000 series fluorescent image analyzer (FujiFilm). The data were processed using Image Gauge v3.41 software (Fuji). The percent of chromosomal fragmentation was calculated as the signal in the lane below the well divided by the combined signal of the lane plus the well and multiplied by 100, as before [15 (link)].