Sapphire Biomolecular Imager

As described previously, the isolated proteins were prepared and separated on 4%‐12% Bis‐Tris Plus Gel (Invitrogen) and transferred to a Polyvinylidene fluoride (PVDF) membrane (iBlot 2 Transfer Stacks, Invitrogen). The membranes were blocked with 2% Bovine Serum Albumin (BSA) in Phosphate Buffered Saline with Tween 20 (PBST) for 1 hour and incubated with antibodies against TLR1, 2, 3, 5, and 6 (1:1000, Novus Biologicals, Littleton, Colorado); TLR4 (1:200, Santa Cruz Biotechnology, Dallas, Texas); and tubulin (1:1000, Abcam, Cambridge, United Kingdom) overnight at 4°C. Thereafter, membranes were washed three times in PBST and further incubated with infrared dye‐conjugated secondary antibodies against mouse (1:7500 for TLR3, 4, or 5) or against rabbit (1:7500 for TLR1, 2, 6, or tubulin). After washing, the blots were visualized using Sapphire Biomolecular Imager (Azure biosystems, Dublin, California) according to the manufacturer's instruction.

A 2038 nt ssDNA was generated by Guide-it™ Long ssDNA Production System (Takara) using oligo 11 and oligo 12 as primers. The exonuclease activities were examined using 15 nM of the 2038 nt ssDNA substrate incubated with 20 nM exonuclease (for EcExoI or EcRecJ) or 0.33 U/μL (for EcExoVII) in 30 μL reaction containing 20 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 125 mM NaCl and 0.1 mM DTT at indicated temperature for 1–30 min. Where indicated, 2 μM EcSSB (Thermo Fisher Scientific) or 500 nM EcUvrD plus 1 mM ATP were included in the exonuclease assay. The reactions were stopped by addition of 25 mM EDTA, 0.2% SDS, 0.67 mg/mL Proteinase K (Denville) and incubated at 50 °C for 1 h. Resulting DNA was resolved on a 1% agarose gel, scanned on a Sapphire Biomolecular Imager (Azure Biosystems) and quantified by ImageQuant software.

Protein (5–10 µg) from the total, mitochondrial, nuclear, and post-mitochondrial extracts from control and treated Rin-5F cells were separated electrophoretically and transferred onto nitrocellulose membrane by Western blotting as described before (Alnahdi et al., 2019a (link); Alnahdi et al., 2020 (link)). Ponceau S staining was used to confirm equal loading of the transferred proteins and then probed with cytochrome c, Bax, Bcl-2, NF-κB p65, I-κB, Cox-2, cytochrome c oxidase (COX), and aconitase antibodies. Immunoreactive proteins were detected by enhanced chemiluminescence using the Sapphire Biomolecular Imager (Azure biosystems, Dublin United States) or using X-ray films. Loading controls used for total/post-mitochondrial, mitochondrial and nuclear fractions were beta-actin, VDAC, and histone H3, respectively. Densitometric analysis was done using Image Studio Lite Ver.5.2 (LI-COR Biosciences, Lincoln, Nebraska, United States) software, and histograms were plotted based on the relative ratios of the proteins normalized to their respective loading control.

This assay was performed using the standard protocol described previously^{24 (link),27 (link)}. Briefly, 25 nM PFV intasome, 50 ng of 3 kb supercoiled (SC) plasmid DNA (pGEM-T Easy, Promega), 10 mM Bis-tris propane, pH 7.5, 110 mM NaCl, 5 mM MgSO₄, 4 μM ZnCl₂, and 10 mM DTT in 15 μl total volume were incubated at 37 °C for 5 min. The reactions were terminated by adding 0.1% SDS, 2.5 mM EDTA, 1 mg/ml proteinase K and incubated at 55 °C for an hour. The products were mixed with 5% glycerol before resolving on a 1% agarose gel in 1X TAE at 105 V for an hour. Gels were stained with 0.1 μg/mL ethidium bromide and scanned on a Sapphire Biomolecular Imager (Azure Biosystems). Unreacted SC plasmid and linear concerted integration products were quantified with AzureSpot software (Azure Biosystems) as described previously^{24 (link),27 (link)} and presented in Supplementary Fig. 1.

Unmodified, recombinant human histones H2A or H2A(K119C), H2B, H3, H3(K56Q), and H4 were expressed and purified as described [19 (link)]. Histones H3(K115ac, K122ac) and H4(K77ac, K79ac) were produced by expressed protein ligation as described [39 (link), 41 (link), 42 (link)]. The synthetic acetylations were confirmed by mass spectrometry analysis (Fig 1). Octamers were refolded at equimolar histone concentrations and purified by Superose 12 10/300 (GE Healthcare) size exclusion chromatography in 10 mM Tris-HCl pH 7.5, 2 M NaCl, 1 mM EDTA. Nucleosomes were reconstituted with Cy5 labelled 145 bp D02 DNA or 147 bp 601 DNA and histone octamer at a 1:1 molar ratio by double dialysis against 5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 mM benzamidine [43 (link)]. The products were separated by sucrose gradient velocity centrifugation [43 (link)]. Gradient fractions were analyzed by separation on a native polyacrylamide gel electrophoresis (PAGE) and imaged using a Typhoon 9410 variable mode fluorescent imager (GE Healthcare) or a Sapphire Biomolecular Imager (Azure Biosystems) (Fig 2). Fractions with fluorescent NPS DNA bound by histone octamer were combined. The sample buffer was exchanged to 5 mM Tris-HCl pH 7.5, 0.5 mM EDTA and nucleosomes were concentrated with Amicon Ultra centrifugal filters (EMD Millipore). Nucleosomes were stored at 4°C.

First-dimension analytical gel electrophoresis was performed, followed by second-dimension sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% fixed concentration gels, as previously described [47 (link),48 (link)]. The gels were scanned with a Sapphire Biomolecular Imager (Azure Biosystems, Dublin, OH, USA) and digitalized via the image analysis software Sapphire Capture system (Azure Biosystems, Dublin, OH, USA). Spot volumes were log-transformed to generate normally distributed data, and log-normalized volume instead of spot intensities was used in statistical processing to quantify differential expression. All spots were pre-filtered and manually checked before applying the statistical criteria (ANOVA test, p ≤ 0.05 and fold ≥ 1.5). Independent direct comparisons were made between the protein spots related to the OB, OD, and ODM groups, and the fold differences and p-values were calculated using one-way ANOVA. Spots that fulfilled the above-mentioned statistical criteria were submitted for further mass spectrometric (MS) analysis.