Cell lines and cell culture<\/p>\n
Bone marrow-derived mouse macrophages were immortalized using the J2 retrovirus, according to the protocol established by Roberson and Walker (1988)74<\/a>. The growth conditions for iBMDMs (kindly provided by Zeinab Abdullah) were Dulbecco\u00b4s modified Eagle\u00b4s medium (DMEM) (Thermo Fisher Scientific, #10566016) supplemented with 10% (v\/v) fetal bovine serum (FBS) (Thermo Fisher Scientific, #10500064) and 100 U\/ml penicillin-streptomycin (Thermo Fisher Scientific, #15140122); the growth conditions for the RAW-Blue NF-\u03baB SEAP reporter cell line (Invivogen, #raw-sp) were the same and in addition 100\u2009\u00b5g\/ml zeocin (InvivoGen, #ant-zn-05), and 100\u2009\u00b5g\/ml normocin (InvivoGen, #ant-nr-05).<\/p>\n If not indicated otherwise, the cells were treated with 25\u2009\u00b5M PDS (Selleckchem, #S7444) and 200\u2009ng\/ml LPS (Sigma, #L7770) or H2O2 (30% (w\/w) Sigma, #H1009) for 4\u2009h or with 10\u2009\u00b5M Nigericin (InvivoGen, #tlrl-nig-5) for 1\u2009h at 37\u2009\u00b0C with 5% CO2.<\/p>\n AlamarBlue assay<\/p>\n The metabolic activity was determined using the alamarBlue Cell Viability Assay Reagent (VWR, #GENO786-921). 60,000 cells were seeded in a black bottom clear 96-well plate and 10\u2009\u00b5l of 10% alamarBlue solution was added (final volume 100\u2009\u00b5l per well) and incubated for 3\u2009h at 37\u2009\u00b0C. The emission at 590\u2009nm was measured using a Fluorskan TM FL plate reader (Thermo Fisher Scientific).<\/p>\n Western blot analysis<\/p>\n Cells were treated with PDS, LPS or both for 4\u2009h. Nigericin was added for 1\u2009h to provide the second signal for full inflammasome activation. For detection of secreted proteins, 1\u2009ml of supernatant was precipitated with trichloroacetic acid, washed with acetone, and the pellet resuspended in 30\u2009\u00b5l L\u00e4mmli buffer. The protein lysates were separated by 10% SDS-PAGEs and blotted on a Protran BA83 Nitrocellulose membrane (GE Healthcare). Blocking was performed with 5% (w\/v) BSA (AppliChem, #A6588) in TBS-T and incubated with the primary antibodies overnight at 4\u2009\u00b0C with agitation. The membranes were washed three times for 5\u2009min with TBS-T, incubated with appropriate fluorescent-coupled secondary antibodies for 1\u2009h at RT and followed by three washing steps. Reactions were detected using the Molecular Imager ChemiDOC XRS Imaging system (Bio-Rad) with automatically calculated detection times by the ChemiDOC software. ImageJ (Fiji75<\/a>) was used to determine the protein intensities.<\/p>\n Used antibodies<\/p>\n Phospho p65 (1:1,000 Cell Signaling, #9936); Donkey anti-rabbit 800 CW (1:10,000 LI-COR, #926-32213); Donkey anti-mouse 800 CW (1:10,000 LI-COR, #926-32212); p65 (1:300 Santa Cruz, #sc8008); Phospho-histone H2A.X (1:1,000 Cell Signaling, #9718); GAPDH (1:1,000 Santa Cruz, #sc69778); histone H3 (1:1,000 Abcam, #Ab1791); IL-1\u03b2 (1:1,000 Proteintech, #26048-1-AP); RelB (1:1,000 Proteintech, #25027-1-AP); Phospho-RelB (1:1,000 Cell Signaling, #5025\u2009T); p105\/p50 (1:1,000 Proteintech, #14220-1-AP); Phospho-p50 (1:200 Santa Cruz, #sc-271908); Caspase-1 (1:1,000 Adipogen, #AG-20B-0042-C100); NLRP3 (1:5,000 Proteintech, #30109-1-AP)<\/p>\n Detection of G4 with BG4 antibody<\/p>\n Immunofluorescence staining of G-quadruplex (G4) structures was performed as previously described76<\/a>. Briefly, immortalized bone marrow-derived macrophages (iBMDMs) were seeded onto glass coverslips in 24-well plates at a density of 7\u2009\u00d7\u2009104 cells per well. Twenty-four hours post-seeding, cells were treated with PDS for 4\u2009h. To preserve G4 structures, cells were pre-fixed using a 1:1 mixture of culture medium (DMEM) and methanol\/acetic acid (3:1) for 5\u2009min at room temperature. Cells were then incubated in methanol\/acetic acid (3:1) for 10\u2009min at room temperature. The fixed cells were permeabilized with 0.1% (v\/v) Triton X-100 in PBS for 3\u2009min. Samples were blocked by incubating the coverslips in blocking solution (2% (w\/v) nonfat dry milk (Carl Roth, #T145) in PBS, pH 7.4) for 1\u2009h at room temperature. For specific G4 detection, samples were incubated with 2\u2009\u00b5g of the FLAG-tagged BG4 antibody per slide in blocking solution for 2\u2009h at room temperature. The primary detection was performed using a rabbit anti-DYKDDDDK epitope antibody (1:800; Cell Signaling Technology, #2368) for 1\u2009h, followed by incubation with a Cyanine 3-conjugated goat anti-rabbit IgG secondary antibody (1:1000; Life Technologies, #A10520) for 1\u2009h at room temperature under gentle rocking. After each incubation step, the cells were washed three times for 10\u2009min with 0.1% (v\/v) Tween-20 (Carl Roth, #9127) in PBS. Coverslips were mounted onto glass slides using Fluoroshield mounting medium containing DAPI (Merck, #F6057) for nuclear counterstaining.<\/p>\n BG-flow<\/p>\n BG-Flow was performed as described in DeMagis et al.76<\/a>. After trypsinization, cells were harvested by centrifugation. Cell pellets were resuspended in 1\u2009ml of a 1:1 mixture of DMEM and methanol\/acetic acid (3:1) for 5\u2009min at room temperature (RT). After centrifugation at 300\u2009g for 5\u2009min at RT, cells were fixed in 3:1 (v\/v) methanol\/acetic acid for 10\u2009min at RT, and then for 15\u2009min at RT. Following two PBS washes, cells were permeabilized with 0.1% (v\/v) Triton X-100 in PBS for 5\u2009min at RT and washed twice with PBS. Cells were blocked using 2% (w\/v) nonfat dry milk in PBS for 45\u2009min at RT. For target detection, cells were sequentially incubated at RT on a tube rotator (30\u2009rpm) with 5\u2009\u03bcg BG4 in blocking buffer for 2\u2009h, followed by a rabbit anti-FLAG antibody (Cell Signaling, #2368; 1:250) for 1\u2009h, and an Alexa Fluor 488 secondary antibody (Invitrogen, #A11008; 1:600) for 1\u2009h. Between antibody incubations, cells were washed twice with 0.1% (v\/v) Tween in PBS. After the final incubation, cells were washed once with 0.1% Tween in PBS and once with standard PBS. Where indicated, DNA was co-stained using 10\u2009\u03bcg\/ml DAPI (alternatively 50\u2009\u03bcg\/ml PI or 1.2\u2009\u03bcg\/ml Hoechst33258) and 50\u2009\u03bcg\/ml RNase A (Ambion, #AM2271) in PBS for 30\u2009min at 37\u2009\u00b0C. Finally, cells were resuspended in 1\u2009ml PBS and analyzed on a FACSCanto II Cell Analyzer (BD Biosciences). Data was processed using FlowJo (BD Biosciences), gating populations by forward scatter (FSC) and side scatter (SSC). Samples omitting the BG4 primary antibody served as negative controls.<\/p>\n CD40 and CD80 expression analysis and mitochondrial fitness<\/p>\n The iBMDMs were collected, washed and resuspended in 50\u2009\u00b5l antibody mix 1) MTG\u2009+\u2009MTDR (Fc-block, 1:100, Invitrogen, #14-0161-86; MTG-FITC, 1:4,000, Invitrogen, #M7514; MTDR-APC, 1:4,000, Invitrogen, #M22426; F4\/80 BV421, 1:200, #Biolegend 123132; L\/D Near-IR, 1:800, Invitrogen, #L34976A); 2) MTG+MitoSox (Fc-block 1:100; MTG-FITC 1:4,000; MitoSox PE, 1:4,000, Invitrogen, #M36008, F4\/80, 1:200; L\/D, 1:800); 3) (Fc-block, 1:100; CD40 PE, 1:200, eBioScience, #12-0401-83; CD80 AF647, 1:200, Biolegend, #104718; F4\/80, 1:200; L\/D, 1:1,000) and proceeded for flow cytometry analysis using a FACS Canto I. Appropriate single stains and FMOs were included. FlowJo software was used for analysis.<\/p>\n RNA isolation<\/p>\n The RNA was isolated using TRIzol (Thermo Fisher Scientific, #15596026) according to the manufacturer\u00b4s instructions.<\/p>\n RNA sequencing<\/p>\n The 3\u00b4RNA sequencing was performed at the NGS core facility Bonn at HiSeq 2500 V4 (10\u2009M raw reads; 1\u00d7150\u2009bp). The analysis was performed using the Partek Flow software (Partek Inc.). Sequencing reads were aligned to mm10 using the STAR aligner.<\/p>\n Standard quantitative PCR<\/p>\n For reverse transcription, the QuantiTect Reverse Transcription Kit (Qiagen, #205311) was used according to the manufacturer\u2019s instructions. The quantification of cDNA was performed using the iQ SYBR Green Supermix (Bio-Rad, #170-8880) on a CFX96 Real Time System with a C1000 Touch Thermal Cycler (Bio-Rad) with the primers in Table\u00a01<\/a>.<\/p>\n Table 1 qPCR primers used for Il1b cDNA quantificationPhagocytosis uptake assay with Salmonella<\/p>\n 1\u00d7106 iBMDMs were mixed with 50\u2009\u00b5l\/ml normal mouse serum and incubated for 15\u2009min at 37\u2009\u00b0C. 2\u00d7108 CFU\/ml mCherry-labeled heat-inactivated Salmonella (provided by the Abdullah lab) were incubated for 2\u2009h at 37\u2009\u00b0C under rotation. The cells were collected, washed with PBS and stained for L\/D (1:800; Near-IR, Invitrogen, #L34976A), CD11b (1:400; FITC, Pharmingen, #553310), F4\/80 (1:400; BV421, #Biolegend 123132), Fc Block (1:100, Invitrogen, #14-0161-86) and incubated for 30\u2009min at 4\u2009\u00b0C. The samples were washed with PBS and proceeded for flow cytometry analysis using a Fortessa II (BD Biosciences). Appropriate single stains and controls without bacteria were included. FlowJo software was used for analysis.<\/p>\n Phagocytosis killing assay with Listeria<\/p>\n 500,000 iBMDMs were seeded (DMEM without antibiotics), if indicated, treated for 20\u2009h with 100\u2009ng\/ml recombinant mouse INF\u03b3 and infected with 50\u2009\u00b5l Listeria (MOI 5). After 30\u2009min the medium was replaced with medium containing gentamycin (100\u2009\u00b5g\/ml) and after 1\u2009h (t\u2009=\u20090) and 4\u2009h (t\u2009=\u20091) the cells were washed twice with PBS and lysed with 500\u2009\u00b5l lysis buffer (0.01% (v\/v) Triton X-100 in ddH2O). Serial dilutions (1:10 dilution was diluted 5 times) with the lysate were performed and plated on BHI plates (20\u2009\u00b5l were plated). The next morning, the colonies were counted, and the CFU\/ml was calculated.<\/p>\n HTRF IL1B<\/p>\n The supernatants of triplicates were collected and the assay performed according to the manufacturer\u2019s instructions (Cisbio, #62MIL1BPEG). In short: 8\u2009\u00b5l sample, 8\u2009\u00b5l medium, 4\u2009\u00b5l antibody mix and 16\u2009\u00b5l standard were used, and a SpectraMax M5e plate reader (Molecular Devices) was used (665 and 620\u2009nm).<\/p>\n QUANTI-Blue assay with RAW NF-\u03baB cells (InvivoGen)<\/p>\n The assay was performed based on the manufacturer\u00b4s instructions. In brief, 100,000 cells per well were seeded in a 96-well plate. 190\u2009\u00b5l QUANTI-BlueTM Solution (InvivoGen, #rep-qbs) were incubated with 10\u2009\u00b5l treated RAW-Blue cell supernatant and incubated at 37\u2009\u00b0C for 6\u2009h. The SEAP levels were measured using a SpectaMax M5e spectrophotometer at 630\u2009nm.<\/p>\n Procartaplex<\/p>\n The supernatants of the iBMDMs were collected in triplicate. The Procartaplex 9 Plex (Thermo Scientific, #D4445144) was used according to the manufacturer\u00b4s instructions. The concentrations were calculated using Thermo Scientific software based on standard curves and measuring the Mean Fluorescence Intensity (MFI).<\/p>\n Inflammasome activation measurement via hASC-mCerulean<\/p>\n Immortalized mouse macrophages with hASC-mCerulean reporter were used for this assay. The well imaging plate (Greiner bio, #6055300) was coated with 0.01% Poly-L-Lysine solution (Sigma-Aldrich, #P8920-100ML) for 15\u2009min at 37\u2009\u00b0C and washed three times with PBS. 50,000 (n\u2009=\u20091) and 30,000 (n\u2009=\u20092, 3) cells per well were seeded. The cells were treated with PDS and\/or LPS and in the last hour with 10\u2009\u00b5M nigericin (Invitrogen, #N1495). In the last 30\u2009min of incubation 50\u2009\u00b5M VX-765 solution (Selleckchem, #S2228) was added to all wells. The fixation and nuclear strain were performed in 4% PFA and DRAQ5 (Thermo Fisher Scientific, #62251) (dilution of 1:5000) for 15\u2009min at RT in the dark. The plate was centrifuged at 340\u2009g for min, the supernatant was removed and replaced with 200\u2009\u00b5l PBS. For analysis, the automated imaging on the Cell Discoverer 7 (Carl Zeiss Microscopy) was used (imaging settings: 10 images per well, 6 z-stack planes spanning 10\u2009\u00b5m with intervals of 2\u2009\u00b5m; Objectives n\u2009=\u20091: Plan-Apochromat 20x\/0.95 with 1x Tubulens (20x magnification); n\u2009=\u20092,3: Plan-Apochromat 5x\/0.35 with 2x Tubulens (10x magnification). The maximum intensity projections from all planes in Zen 2.6 (blue edition, Carl Zeiss Microscopy) software were generated, and the bright field channel was removed. The new file was used for automated quantification in the CellProfiler (3.1.9) Software.<\/p>\n ATAC sequencingSample preparation and sequencing<\/p>\n For ATAC-Seq, 50,000 cells per condition were washed with PBS and lysed in 50\u2009\u00b5l of lysis buffer (10\u2009mM Tris-HCl, pH 7.4, 10\u2009mM NaCl, 3\u2009mM MgCl2, 0.1% (v\/v) NP-40). Following centrifugation (500\u2009g for 10\u2009min at 4\u2009\u00b0C), the nuclear pellet was resuspended in a mix comprising 25\u2009\u00b5l of 2x TD buffer, 2.5\u2009\u00b5l of Tn5 transposase (Nextera DNA Library Kit, Illumina, #FC-121-1030), and 22.5\u2009\u00b5l of nuclease-free H2O. The reaction was incubated for 30\u2009min at 37\u2009\u00b0C. DNA was subsequently purified using the MinElute Reaction Cleanup Kit (Qiagen, #28204) according to the manufacturer\u2019s instructions and eluted in 10\u2009\u00b5l of elution buffer. Sequencing was performed at the Cologne Center for Genomics on an Illumina platform, using 50 million 150-bp paired-end reads per sample.<\/p>\n Data processing and peak calling<\/p>\n Sequence read preprocessing and alignment were performed using the following pipeline. Raw reads were trimmed using Cutadapt (v1.18)77<\/a> and aligned to the mouse reference genome (mm10) using Bowtie2 (v2.4.5)78<\/a>. Alignments were sorted with SAMtools (v1.14)79<\/a> and PCR duplicates were removed using Picard (v2.26.10) (https:\/\/github.com\/broadinstitute\/picard<\/a>). Peak calling on the sorted and filtered BAM files was performed using MACS2 (v2.2.7.1)80<\/a>. To ensure high reproducibility across replicates, consensus peaks for each condition were defined using the Irreproducible Discovery Rate (IDR) framework (v2.0.4.2)81<\/a>. Differentially accessible regions between conditions were identified utilizing the DiffBind package (v3.10) (http:\/\/bioconductor.org\/packages\/release\/bioc\/html\/DiffBind.html<\/a>).<\/p>\n Fast ChIP<\/p>\n Chromatin immunoprecipitation was performed using the FastChIP protocol established by Bomsztyk and colleagues82<\/a>. Briefly, iBMDMs were plated at a density of 6\u20138\u2009\u00d7\u2009106 cells in 10\u2009cm dishes. Cells were washed and covered with 5\u2009ml PBS before crosslinking with 1.42% formaldehyde (Carl Roth, #4980) for 15\u2009min at room temperature. The reaction was quenched with 125\u2009mM glycine for 5\u2009min at room temperature, and cells were washed twice with cold PBS, scraped into 1\u2009ml PBS, and pelleted at 2000 g for 5\u2009min at 4\u2009\u00b0C. Pellets were resuspended in 1\u2009ml IP buffer (150\u2009mM NaCl, 50\u2009mM Tris-HCl, pH 7.5, 5\u2009mM EDTA, 0.5% NP-40, and 1% Triton X-100, protease inhibitors), and lysed by pipetting. After centrifugation at 12,000\u2009\u00d7\u2009g for 1\u2009min at 4\u2009\u00b0C, the pellet was washed once with 1\u2009ml IP buffer and centrifuged again. For sonication, pellets were resuspended in 1\u2009ml IP buffer per 10 million cells, split into 300\u2009\u00b5l aliquots, and sheared using a Bioruptor (Diagenode) for five cycles of 30\u2009s on\/30\u2009s off, yielding average fragment sizes of 300\u2013600\u2009bp. Lysates were clarified at 12,000\u2009\u00d7\u2009g for 10\u2009min at 4\u2009\u00b0C, and chromatin aliquots equivalent to 2 million cells were incubated overnight at 4\u2009\u00b0C with a 1:100 dilution of a rabbit anti-NF-\u03baB p65 antibody (D14E12, Cell Signaling Technology, #8242S) or an equivalent amount of a rabbit IgG control (Thermo Fisher Scientific, #02-6102. The next day, samples were centrifuged at 12000\u2009g for 10\u2009min at 4\u2009\u00b0C, and the supernatant was then incubated with 20\u2009\u00b5l of pre-washed protein G-Dynabeads (Invitrogen, #10446293) for 45\u2009min at 4\u2009\u00b0C. Beads were washed five times with 1\u2009ml cold IP buffer and resuspended in 100\u2009\u00b5l Proteinase K buffer (100\u2009mM Tris-HCl, pH 7.5, 150\u2009mM NaCl, 12.5\u2009mM EDTA, 2% SDS, and 5\u2009mM DTT). Samples were treated with 1\u2009\u00b5l of 20\u2009mg\/ml Proteinase K (Carl Roth, #3719) for 30\u2009min at 55\u2009\u00b0C, followed by boiling at 95\u2009\u00b0C for 10-15\u2009min to inactivate the enzyme and decrosslink the chromatin. DNA was precipitated with 300\u2009\u00b5l ethanol and 15\u2009\u00b5g GlycoBlue (Thermo Fisher Scientific, #AM9515) at \u221220\u2009\u00b0C for at least 1\u2009h, pelleted at 20000\u2009g for 30\u2009min at 4\u2009\u00b0C, washed with 70% ethanol, and resuspended in 50\u2009\u00b5l water. qPCR was performed with 2\u2009\u00b5l of the eluted DNA with the iQ SYBR Green Supermix (BioRad) using the primers in Table\u00a02<\/a>.<\/p>\n Table 2 qPCR primers used for Fast ChIPAnalysis of NF-\u03baB binding sites and G4s in the Il1b promoter<\/p>\n We analyzed the DNA sequence for NF-\u03baB binding sites using position weight matrices (PWMs) from the HOCOMOCO v11 (https:\/\/hocomoco14.autosome.org\/<\/a>) database (RELA, RELB, REL, NF\u039aB1, NF\u039aB2) and TFBIND<\/a>. Both DNA strands were scanned, and for each subsequence, the raw PWM score was normalized to a relative score between 0 and 1 using the theoretical minimum and maximum possible scores for that PWM. Sites with a relative score \u2265 0.80 were retained. To avoid redundancy, predicted sites located within 5\u2009bp of each other were merged by keeping the highest-scoring site in the cluster. G4s were predicted by G4RNA screener83<\/a> using the algorithms cGcG, G4hunter and G4NN with a window size of 30 and step size of 5. Running scores are reported for each step.<\/p>\n G4-DNA precipitation (G4DP)<\/p>\n For pulldown of chromatin G4s, a modified variant of the G4DP method established by Feng et al. (2022)51<\/a> was employed. Briefly, iBMDMs were seeded at 6 to 8 million cells in 10\u2009cm dishes. The following day, cells were washed, overlaid with 5\u2009ml PBS, and crosslinked using 1% formaldehyde for 10\u2009min at room temperature. Crosslinking was quenched with 125\u2009mM glycine for 5\u2009min at room temperature, followed by two cold PBS washes, scraping into 1\u2009ml cold PBS, and pelleting at 2000 g for 5\u2009min at 4\u2009\u00b0C. Cell pellets were lysed in 1\u2009ml cold nuclear isolation buffer (10\u2009mM Tris-HCl pH 7.4, 10\u2009mM NaCl, 3\u2009mM MgCl2, 0.1% NP-40, 1\u2009mM DTT, PMSF) by pipetting and 15\u2009min incubation on ice. After centrifugation at 1000\u2009g for 5\u2009min at 4\u2009\u00b0C, nuclear pellets were resuspended in 300\u2009\u00b5l sonication buffer (10\u2009mM Tris-HCl, pH 7.4, 5\u2009mM EDTA, 150\u2009mM KCl, 0.5% NP-40, 0.5% SDS, 0.5\u2009mM DTT, PMSF). Chromatin was sheared using a Bioruptor for 10 cycles of 30\u2009s on\/30\u2009s off to generate fragments of 100-300\u2009bp, clarified at 12000\u2009g for 10\u2009min at 4\u2009\u00b0C, and diluted with 1.2\u2009ml incubation buffer to reduce the SDS concentration below 0.1%. The lysate was treated with 15\u2009\u00b5l of 10\u2009mg\/ml RNase A for 30\u2009min at 37\u2009\u00b0C with shaking. 10% of the lysate was reserved as the input sample. For the pulldown, chromatin aliquots equivalent to ~2 million cells were incubated overnight at 4\u2009\u00b0C with 100\u2009\u00b5M BioCyTASQ (Merck, #SCT246). Streptavidin Dynabeads M280 (Invitrogen, #10202983) were washed with incubation buffer, added to the sample, and rotated for 4\u2009h at 4\u2009\u00b0C. To reduce genomic background, beads were washed twice with 1\u00d7 rCutSmart buffer (NEB, #B6004S), resuspended in 100\u2009\u00b5l of the same buffer with 2 U double-strand-specific dsDNase (Sigma Aldrich, #EN771), and incubated for 10\u2009min at 37\u2009\u00b0C with shaking, followed by three washes with cold incubation buffer. DNA was eluted twice using 200\u2009\u00b5l elution buffer containing 100\u2009mM NaHCO3 and 1% SDS at 65\u2009\u00b0C for 15\u2009min, pooled, supplemented with 16\u2009\u00b5l 5\u2009M NaCl, and decrosslinked for 4\u2009h to overnight at 65\u2009\u00b0C. DNA was extracted using phenol-chloroform-isoamyl alcohol, precipitated overnight at \u221220\u2009\u00b0C with 15\u2009\u00b5g glycoblue and 1\u2009ml ethanol, washed with 70% ethanol until SDS was dissolved, and eluted in 100\u2009\u00b5l water. qPCR was performed with 2\u2009\u00b5l of the eluted DNA with the iQ SYBR Green Supermix (BioRad) using the primers in Table\u00a03<\/a>.<\/p>\n Table 3 qPCR primers used for G4DPGeneration of nuclear lysates from iBMDMs<\/p>\n Nuclear extracts were prepared utilizing an optimized rapid fractionation protocol84<\/a>. Briefly, 5\u2009\u00d7\u2009106 cells were harvested, washed with 1\u2009ml of ice-cold PBS, and pelleted. The cell pellet was resuspended in 5 packed cell volumes of hypo-osmotic lysis buffer (0.3\u2009M sucrose, 2% Tween-40, 10\u2009mM HEPES-KOH pH 7.9, 10\u2009mM KCl, 1.5\u2009mM MgCl2, 0.1\u2009mM EDTA, 0.5\u2009mM DTT, protease inhibitors). To release intact nuclei, the suspension was subjected to mechanical disruption via repeated pipetting (\u2009~\u2009100 times). The resulting lysate was carefully layered over a 1.5\u2009M sucrose cushion (1.5\u2009M sucrose, 10\u2009mM HEPES-KOH pH 7.9, 10\u2009mM KCl, 1.5\u2009mM MgCl2, 0.1\u2009mM EDTA, 0.5\u2009mM DTT, and protease inhibitors) and centrifuged for 10\u2009min to isolate the nuclear fraction. The nuclear pellet was washed with low salt wash buffer (10\u2009mM HEPES-KOH pH 7.9, 10\u2009mM KCl, 1.5\u2009mM MgCl2, 0.1\u2009mM EDTA, 0.5\u2009mM DTT, and protease inhibitors) and subsequently extracted in 100\u2009\u00b5l of high salt extraction buffer (20\u2009mM HEPES-KOH pH 7.9, 420\u2009mM NaCl, 1.5\u2009mM MgCl2, 0.2\u2009mM EDTA, 25% glycerol, 0.5\u2009mM DTT, and protease inhibitors) for 20\u2009min on ice with intermittent gentle vortexing. The extract was cleared via centrifugation, and the supernatant containing high-purity nuclear proteins was collected, aliquoted, and stored at \u221280\u2009\u00b0C.<\/p>\n Electrophoretic mobility shift assay (EMSA)Oligonucleotide hybridization and G-quadruplex folding<\/p>\n Fluorescently labeled oligonucleotides were designed to contain a central G-quadruplex (G4)-forming motif flanked by duplex-forming genomic sequences. To facilitate structured G4 folding within the flanking double-stranded context and to mimic the transient single-stranded state during transcriptional activation, a specialized denaturation-renaturation sequence was applied. Sense and antisense strands were diluted to a working concentration of 0.6\u2009\u00b5M in binding buffer (10\u2009mM Tris\u2013HCl pH 7.4, 1\u2009mM EDTA, 150\u2009mM KCl, and 5% glycerol). The mixtures were denatured, flash-cooled on ice, and subsequently allowed to fold and anneal at 37\u2009\u00b0C in the presence or absence of 25\u2009\u00b5M PDS. Untreated control duplexes and single-stranded references were prepared under identical conditions. As a positive control, a double-stranded oligo containing an NF-\u03baB consensus sequence (Cy5-GTTGAGGGGACTTTCCCAGGCA) that does not fold into a G4. This sequence was also used as an unlabeled competitor oligo to validate the NF-\u03baB-dependency of shifted bands.<\/p>\n