HEK293T, HeLa, U937, and K562 cells were obtained from ATCC. HEK293T and HeLa were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, LONSERA). U937 and K562 cells were cultured in RPMI-1640 medium (Gibco) with 10% FBS. MDA-231-LM2 (LM2) cells were kindly provided by Dr. Yibin Kang and grown in DMEM supplemented with 10% FBS. Drosophila S2 cells were obtained from Invitrogen (R690-07) and maintained in Schneider’s medium. Mouse embryonic fibroblast (MEF) cells were grown in DMEM with 10% FBS. All mammalian cells were cultured at 37°C with 5% CO2. Cells were confirmed to be mycoplasma negative using MycoBlue Mycoplasma Detector (Vazyme, D101-01). Live cells were quantified using a TC20 automated cell counter (Bio-Rad).
Chemicals and antibodies
δ-Aminolevulinic acid hydrochloride (ALA; CAS: 5451-09-2), hemin (CAS: 16009-13-5), protoporphyrin IX (PpIX; CAS: 553-12-8), biotinyl tyramide (CAS: 41994-02-9), and succinylacetone (SA; CAS: 51568-18-4) were obtained from MedChemExpress (MCE) and dissolved in H2O, 0.1 M NaOH, and dimethyl sulfoxide (DMSO; Sigma-Aldrich), respectively, to make stock solution. Pyridostatin trihydrochloride (PDS; CAS: T4457) was purchased from Topscience Co., Ltd. Pol II Rpb1 (POLR2A) NTD (D8L4Y) rabbit mAb, Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb, and TFIIB (GTF2B, 2F6A3H4) mouse mAb were purchased from Cell Signaling Technology. TBP mouse mAb (66166-1-Ig), BACH1 polyclonal antibody (14018-1-AP), and NRF2/NFE2L2 polyclonal antibody (16396-1-AP) were purchased from Proteintech. FLAG-synthetic antibody (M2) was obtained from Sigma-Aldrich. Biotin rabbit mAb (A20684), EP300 rabbit pAb (A13016), RBBP5 rabbit pAb (A6965), and mouse anti-His tag antibodies (AE003) were purchased from ABclonal. HMOX1 Rabbit pAb (380753) was obtained from Zen-bioscience. Homemade histone H3 Lysine 4 methylation antibodies were generated as previously described . The scFv antibodies BG4 and BG4-EGFP were purified using the pSANG10 expression vector according to the published protocols [21, 56]. The specificity of purified BG4 and BG4-EGFP to G4 has been confirmed in a previous study . Recombinant pA-Tn5 purification and pA-Tn5 transposome assembly were performed as described previously .
Hemin and PpIX extraction from cells and analysis with LC-ESI-MS/MS
Hemin and PpIX were extracted from cells according to a previous study  with some modifications. Briefly, twenty million cells were collected, resuspended with 300 μL of TE buffer (10 mM Tris-HCl, 1mM EDTA at pH 7.2), and shaken for 1 h at room temperature. Samples were pre-chilled on ice and cells were efficiently disrupted with ultrasonication. Nine hundred microliters of acetonitrile was added to the sample with a 5-min incubation on a shaker at room temperature. After centrifugation at 2500g for 5 min, the supernatant-containing free porphyrin fraction was collected and analyzed for the amount of free PpIX and hemin with liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Next, 800 μL of acetonitrile: 1.7 M HCl (8:2, v/v) was added to the pellet and shaken for 40 min at room temperature to extract PpIX and heme from the protein and chromatin. Two hundred microliters of saturated MgSO4 and 0.1 g of NaCl were added, and shaken for another 5 min. After centrifugation at 2500g for 5 min, the top organic layer was collected to analyze the content of protein-bound and chromatin-bound PpIX and hemin with LC-ESI-MS/MS.
LC-ESI-MS/MS experiments were performed on a Thermo TSQ Quantis triple-stage quadrupole mass spectrometer under the positive ion mode. Injections were automatically performed using an UltiMate 3000 HPLC equipped with an autosampler. A Hypersil GOLD C18 HPLC column was used for chromatographic separation at 40°C. The electrospray ionization was set at 3.5 kV, and the vaporizer temperature was set at 275°C. Selected reaction monitoring (SRM) mode was used, and the cycle time is 0.3 s. The mobile phase was composed of 0.1% formic acid H2O (A) and acetonitrile (B). The gradient condition was used: 0–0.5 min, 5% B; 0.5–5 min, 5–95% B; 5–7 min, 95% B; 7–8.1 min 95–5% B, 8.1–12 min 5% B. The flow rate was 0.2 mL/min. A volume of 5–10 μL of each sample was loaded for each analysis. The precursor m/z for PpIX was 564.2 and the product m/z were 432.28, 490.22, and 505.19 with collision energies of 50.7, 41.86, and 40.93 eV, respectively. Hemin has a precursor m/z of 616 and product m/z of 483.18, 498.14, and 557.21 with collision energies of 55, 52.05, and 38.82 eV.
Bio-layer interferometry assay
Bio-layer interferometry assays were performed using a GatorPrime with Small Molecule, Antibody, and Protein (SMAP) Probes (Gator Bio, 160011), which are high-capacity streptavidin biosensors. Biotin-labeled ssDNA oligonucleotide (5′-[Biotin] GGC ATA GTG CGT GGG CG-3′) and its reverse complement DNA oligo were synthesized by Sangon Biotech and annealed to form biotin-labeled single-stranded and double-stranded DNA. Biotin-labeled MYC-G4 oligonucleotide (5′-[Biotin] TGA GGG TGG GTA GGG TGG GTA A-3′), SPB1-G4 oligonucleotide (5′-[Biotin] GGC GAG GGG CGT GGC CGG C-3′), hTELO-G4 oligonucleotide (5′-[Biotin] GGT TAG GGT TAG GGT TAG GGT TAG GGT TAG-3′), and intermolecular hTELO-G4 oligonucleotide (5′-[Biotin] TTA GGG TTA GGG TTA GGG-3′) were synthesized and annealed to form biotin-labeled parallel, antiparallel, mixed parallel/antiparallel, and intermolecular G4 structures. Kinetic titration series were performed in the interaction buffer (PBS with 0.5% DMSO and 0.02% Tween-20). PpIX and hemin were serially diluted with the interaction buffer from 20 to 1.25 μM and 8 to 0.5 μM, respectively. The SMAP biosensors were hydrated in the interaction buffer for 10 min at 25 °C. Following the initial 120-s baseline, the SMAP biosensors were loaded with the biotin-labeled G4 for 300 s. Redundant probes were removed by a 120-s baseline adjustment. To measure the interaction between porphyrins and G4, the duration time of association and dissociation were set to 120 s for PpIX and 300 s for hemin. One biosensor recorded the kinetic titration series by sequentially interacting with increasing concentrations of porphyrins, with dissociation between each sample concentration. Meanwhile, one sensor recorded the buffer reference signal and two sensors were used as sensor reference. Sensorgrams and sensor signals were analyzed by the Gator Part11 Software.
Synthesis and purification of Biotin-PEG4-hemin
Synthesis and purification of biotin-PEG4-hemin were performed as previously reported  with minor modifications. Hemin and biotin-PEG4-hydrazide (CAS: 756525-97-0, Aladdin) were dissolved in DMF and DMSO at 4.4 and 68 mg/mL, respectively. A volume of 250 μL of hemin was mixed with 5 μL of biotin-PEG4-hydrazide and 1.4 mg of DCC (CAS: 538-75-0, Aladdin). Then the mixture was incubated on a shaker in the dark for 3 h at room temperature. Next, 5% pyridine (CAS: 110-86-1, Aladdin) was added to the sample and the mixture was applied to a CDS Empore C18 Extraction Disks. The biotin-PEG4-hemin was eluted with a gradient of 40–90% acetonitrile in the presence of 0.1% TFA. After vacuum freeze-drying, the sample was dissolved in a small amount of DMSO and stored at −80°C. LC-ESI-MS/MS was used to assay the synthesis of biotin-PEG4-hemin. The precursor m/z for biotin-PEG4-hemin was 1103.45, and the product m/z were 616.25 and 666.22 with collision energies of 55 eV for both.
In situ profiling of hemin binding sites in the genome using biotin-PEG4-hemin-based capture sequencing
Biotin-PEG4-hemin was combined with the Tn5-based CUT&Tag strategy to identify hemin binding sites in the genome. Briefly, one million HEK293T or HeLa cells were resuspended in NE1 buffer (20 mM HEPES at pH 7.9, 10 mM KCl, 0.5 mM spermidine, 0.1% Triton X-100, 20% Glycerol, 1× protease inhibitors) and incubated on ice for 10 min. After centrifugation at 1300g for 10 min, nuclei were isolated and collected. These nuclei were washed once with PBS and resuspended with 100 μL wash buffer (20 mM HEPES at pH 7.5, 150 mM KCl, 0.5 mM spermidine, 1× protease inhibitors). Ten microliters pre-activated concanavalin A-coated magnetic beads (Smart-Lifesciences) were added to these nuclei and incubated on a rotator for 10 min at room temperature. The supernatant was removed, and bead-bound nuclei were resuspended in 100 μL dig-wash buffer (0.05% digitonin, 20 mM HEPES at pH 7.5, 150 mM KCl, 0.5 mM spermidine, 1× protease inhibitors) containing 2 mM EDTA. Biotin-PEG4-hemin was added to the sample to a final concentration of 3 μM and rotated overnight at 4°C in the dark. To verify the signal specificity, Biotin-PEG4-hemin was competed with 20 μM TMPyP4. These nuclei were washed three times in 200 μL dig-wash buffer to remove unbound biotin-PEG4-hemin. Next, anti-biotin rabbit mAb was diluted 100-fold in 100 μL dig-wash buffer with 2 mM EDTA and was added to the nuclei with 1-h rotation at room temperature. For the control sample, IgG was used to replace anti-biotin antibody. After washing once with dig-wash buffer, these nuclei were resuspended with 100 μL dig-wash buffer with 2 mM EDTA and 1 μL of mouse anti-rabbit IgG, incubated for another hour at room temperature, and further washed three times with dig-wash buffer. A 1:250 dilution of pA-Tn5 adapter complex (~0.2 μM) was prepared in 100 μL dig-300 buffer (0.05% digitonin, 20 mM HEPES at pH 7.5, 300 mM KCl, 0.5 mM spermidine, 1× protease inhibitors) and added to the nuclei followed by 1-h rotation at room temperature in the dark. After five washes with dig-300 buffer, the bead-bound nuclei were resuspended with 40 μL of tagmentation buffer (10 mM TAPS-NaOH at pH 8.5, 10 mM MgCl2, 7.5% DMF) and incubated at 37°C for 1 h. To terminate the tagmentation reaction, 1.5 μL of 0.5 M EDTA, 0.5 μL of 10% SDS, and 1 μL of 20 mg/mL proteinase K were added and incubated at 55°C for 1 h and then for 15 min at 75°C. The DNA was extracted with Sera-Mag carboxylate-modified magnetic beads (GE Healthcare) for library preparation. Twenty-one microliters DNA was mixed with a universal i5 and a uniquely barcoded i7 primer and amplified with NEB Q5 high-fidelity 2× master mix. The libraries were purified with 0.56–0.85× volume of Sera-Mag carboxylate-modified magnetic beads and subjected to Bioanalyzer DNA analysis and Illumina sequencing.
Reads were aligned to human (UCSC hg38) genome with Bowtie2 version 2.2.6 , using parameters: --local --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700. The aligned reads were normalized with total reads aligned (reads per million, r.p.m.). The track files were made with the bamCoverage command from deeptools 3.3.1 . Peaks were called using the IgG controls and MACS2 version 2.1.2  with default parameters and a p-value cutoff of 1E−7. The distribution of hemin binding peaks was annotated with ChIPpeakAnno. To perform the motif analysis, the summits of the called hemin binding peaks were extended to 50 bp to fetch the DNA sequences, and MEME-ChIP  was used. Heatmaps were made for the indicated windows using the average coverage (r.p.m.).
Genome-wide mapping of endogenous G4-hemin complexes using a G4 self-biotinylation reaction
Identification of endogenous G4-hemin binding sites in the genome was performed using a previously reported self-biotinylation reaction  in combination with the Tn5-based CUT&Tag strategy. Briefly, HEK293T cells were treated with or without 80 μM Hemin for 2 h. To verify the signal specificity, hemin was displaced with 80 μM TMPyP4 or PDS  for another 2 h. The nuclei were isolated from one million cells and resuspended with 100 μL dig-wash buffer (0.05% digitonin, 20 mM HEPES at pH 7.5, 150 mM KCl, 0.5 mM spermidine, 1× protease inhibitors) containing 1 μL of 1 mg/mL RNase A and 3 mM biotin tyramide. Ten millimolars H2O2 was added, and the reaction was performed for 3 min on a shaker at room temperature . The reaction was quenched with PBS solution containing 5 mM Trolox and 10 mM sodium ascorbate and washed with wash buffer (20 mM HEPES at pH 7.5, 150 mM KCl, 0.5 mM spermidine, 1× protease inhibitors). The nuclei were resuspended with 100 μL wash buffer and conjugated to 10 μL pre-activated concanavalin A-coated magnetic beads (Smart-Lifesciences). The bead-bound nuclei were washed three times with dig-wash buffer to remove redundant biotin tyramide and incubated with 50 μL dig-wash buffer containing 2 mM EDTA and 0.5μL of anti-biotin rabbit mAb overnight at 4°C. After washing once, these nuclei were resuspended with 100 μL dig-wash buffer with 2 mM EDTA and 1 μL of rabbit anti-mouse IgG and incubated for another hour at room temperature. The nuclei were further washed three times with dig-wash buffer and tagmented with the pA-Tn5 adapter complexes. The tagmented DNA was used for library preparation and Illumina sequencing. The CUT&Tag sequencing reads were aligned to the human genome (UCSC hg38) using Bowtie2 version 2.2.6  with parameters: --local --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700. The reads were further normalized with total reads aligned (reads per million, r.p.m.). Peaks were called using the IgG controls and MACS2 version 2.1.2  with default parameters and a p-value cutoff of 1E−7. Heatmaps were made for the indicated windows using the average coverage.
G4-CUT&Tag and R-loop CUT&Tag
G4-CUT&Tag and R-loop CUT&Tag were performed as previously described [21, 61].
Briefly, one million cells were resuspended with NE1 buffer by gentle pipetting and incubated on ice for 10 min. The nuclei were isolated and conjugated to 10 μL pre-activated concanavalin A-coated magnetic beads (Smart-Lifesciences). The bead-bound nuclei were resuspended in 50 μL of dig-wash buffer and incubated with 5 μL 0.1 mg/mL BG4 primary antibody or 2 μg recombinant GST-His6-2×HBD protein. For the control samples, IgG was used to replace the BG4 antibody or GST-His6-2×HBD. After overnight incubation at 4°C, the liquid was removed and the bead-bound nuclei were washed once with dig-wash buffer. The Anti-Flag M2 or anti-HisTag antibody (1:100 dilution) were used to bind with BG4 primary antibody or GST-His6-2×HBD. The nuclei were washed with dig-wash buffer and the Rabbit anti-mouse IgG were used to bind with the Anti-Flag M2 or anti-HisTag antibody. Bead-bound nuclei were briefly washed three times with 200 μL of dig-wash buffer and tagmented with pA-Tn5 adapter complex for library preparation [21, 58].
G4-CUT&Tag and R-loop CUT&Tag reads were aligned to the human genome (UCSC hg38) and Escherichia coli genome with Bowtie2 version 2.2.6 , using parameters: --local --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700. The aligned reads were normalized with total reads aligned (reads per million, r.p.m.) and spiked-in E. coli reads. The track files were made with the bamCoverage command from deeptools 3.3.0 . Peaks were called using MACS2 version 2.1.2 with default parameters , and a p-value cutoff of 1E−5. Heatmaps were made for the indicated windows using the average coverage.
Chromatin immunoprecipitation sequencing with reference exogenous genome (ChIP-Rx)
ChIP-Rx was performed with 1×107 human cells and 1×106 MEF cells for spike-in normalization as described in a previous study . For immunoprecipitation, sonicated chromatin was incubated with 10 μg of specific antibodies and 15 μL of pre-blocked Protein A/G beads (Smart-Lifesciences). After extensive washes, the captured DNA was eluted for library preparation using the NEBNext Ultra II DNA library prep kit for Illumina before sequencing on a NovaSeq 6000. ChIP-Rx reads were aligned to the human genome (UCSC hg38) and mouse genome (mm10) with Bowtie2 version 2.2.6 , using parameters: --local --very-sensitive. The resulting reads were normalized with the aligned mouse reads. The aligned human BAM files were normalized and converted to bigwig files for visualization in the UCSC Genome Browser. Peaks were called using MACS2 version 2.1.2 with default parameters . MEME-ChIP  was used to further analyze peak region motifs. Heatmap and metaplots were made for the indicated windows using the average coverage (r.p.m.).
Quick precision run-on sequencing (qPRO-seq)
qPRO-seq was performed as previously reported [21, 62], and qPRO-seq libraries were sequenced on the NovaSeq 6000 platform. qPRO-seq reads were aligned to the human hg38 genome with Bowtie version 1.1.2 . UMI-tools was used to extract UMIs and remove duplications . The resulting reads were normalized to total reads used for alignment (reads per million, r.p.m.) and converted to bigwig files for visualization in the UCSC Genome Browser. Heatmap and metaplots were made for the indicated windows using the average coverage (r.p.m.).
mRNA sequencing (mRNA-seq)
Following hemin treatment for 6 h, cells were collected and lysed with Trizol reagent. Total RNA was extracted according to the manufacturer’s instructions. One microgram total RNA was spike-in with 100 ng Drosophila S2 RNA and was further used for mRNA isolation with VAHTS mRNA Capture Beads (Vazyme, N401) following the manufacturer’s instructions. Library preparation was performed with the NEBNext Ultra II Directional RNA Library Prep Kit, followed by sequencing on a NovaSeq 6000. RNA-seq reads were aligned to the human genome (UCSC hg38) or mouse genome (mm10). Alignments were processed with HISAT2 , allowing only uniquely mapping reads with up to three mismatches within the 150-bp reads. The read counts across each gene were counted with featureCounts version 2.0.0, and DESeq2 was used to perform the differential gene expression analysis . To estimate the fold changes based on the Drosophila spike-in RNA, size factors were calculated on the counts of the Drosophila genes and applied to the human gene counts before fold change estimation with DESeq2. Heatmaps were generated using R package 3.4.3 with normalized counts from DESeq2. Gene ontology enrichment was analyzed with Metascape  for upregulated and downregulated genes in response to hemin treatment.
Immunofluorescence microscopy and analysis
HeLa cells or mouse primary hepatocytes were seeded on a coverslip and cultured for 24 h before treatment. Cells were then fixed with pre-chilled methanol for 10 min. After three washes with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS buffer for 15 min and blocked with 8% BSA in PBS for 1 h. Next, cells were incubated with 2 μg recombinant BG4-EGFP antibody in 2% BSA for 2 h and washed five times for 1 h with 0.1% Tween 20 in PBS under gentle rocking. For nuclear staining, cells were incubated with 2 μg/μL of DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) (Thermo Fisher Scientific, D1306) for 15 min. After mounting, slides were imaged with a Leica SP8 microscope. EGFP was excited by the 488-nm laser line, and the fluorescence was detected in the range of 498–545 nm. The 405-nm laser line was used to excite DAPI, and the emission between 413 and 462 nm was collected. For each group, images of about 6 fields of vision were taken for every sample without Z-stack under fixed laser line intensity. For quantification of G4 intensities, the same number of nuclei in each group were randomly selected to calculate the mean fluorescence intensities of nuclear G4s by dividing the total intensities of BG4-EGFP with DAPI-marked nuclear area using the Leica LASX software. The same set of samples was imaged and processed in one batch to reduce experimental variation. The G4 staining profile of HeLa cells is available for download from figshare repository (https://doi.org/10.6084/m9.figshare.21608112.v1  and https://doi.org/10.6084/m9.figshare.21608100.v1 ).
Reporter gene assays
Putative G4-forming regions in the promoters of NPM1, SRSF4, HSPA8, H2AX, and SERBP1, showing signal enrichments in G4-CUT&Tag, were inserted into the pGL3 basic luciferase reporter vector (Promega). HEK293T cells were seeded in 24-well plates and transfected with or without these recombinant plasmids. Two hours post-transfection, ALA, PpIX, or hemin were added to the medium. Then the cells were cultured for another 24 h before assaying for luciferase activity with the firefly luciferase assay kit (US Everbright Inc.) following the manufacturer’s instructions.
Quantitative RT-PCR analysis
Cells were lysed with Trizol reagent, and total RNA was extracted according to the manufacturer’s instructions. Reverse transcription was performed using the ReverTra Ace qPCR RT Master Mix from Toyobo life science (FSQ-301). Real-time PCR was performed with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02). Samples were amplified with primers for murine Hmox1 gene (forward: 5′-AAG CCG AGA ATG CTG AGT TCA-3′; reverse: 5′-GCC GTG TAG ATA TGG TAC AAG GA-3′) and 18S rRNA (forward: 5′-TGT GCC GCT AGA GGT GAA ATT-3′; reverse: 5′-TGG CAA ATG CTT TCG CTT T -3′) as a reference gene. The cycle threshold Ct values were normalized to the 18S rRNA curve. PCR experiments were performed in triplicate and standard deviations were calculated and displayed as error bars.
Isolation and culture of mouse primary hepatocytes
Isolation and culture of mouse primary hepatocytes were performed using a two-step perfusion method as previously published [69, 70] with some modifications. Eight-week-old C57BL/6 mice were used in this experiment. The anesthetized mice were placed on the dissection trap, and their limbs were secured using needles. A “U”-shaped incision was made through the skin, and both the portal vein and vena cava were exposed. Then the inferior vena cava was cannulated, and the liver was perfused with 20 mL of EGTA-containing buffer-I (2 mM glutamine, 0.5% glucose, 25 mM HEPES, 2 mM EGTA diluted in PBS) to wash out the blood and circulating cells from the liver as well as to eliminate calcium via EGTA. Next, 0.3 mg/mL collagenase type IV (Sigma-Aldrich) in Ca2+-containing buffer-II (2 mM glutamine, 0.5% glucose, 25 mM HEPES, 3 mM CaCl2 diluted in Williams E medium) was perfused to the liver to digest collagen in the extracellular matrix, thereby facilitating cell dispersion. The liver was gently dissected and transferred to a 10-cm plate. Collagenase-containing Ca2+-containing buffer-II was added to the plate, and the liver was ruptured by fine tip forceps and gentle pipetting. The suspension was filtered through a 70-μm cell strainer and ice-cold Ca2+-containing buffer-II was used to rinse the plate and added to the cell strainer. After centrifugation at 4°C, 50g for 3 min, the supernatant was removed and the hepatocyte-containing pellet was washed three times with ice-cold Ca2+-containing buffer-II. The isolated hepatocytes were resuspended with Williams E medium supplemented with 10% fetal bovine serum, 50 U/mL penicillin, 50 μg/mL streptomycin, 5 μg/mL insulin, and 50 μM hydrocortisone hemisuccinate. After counting living cells, these hepatocytes were plated on collagen-coated cell culture plates/wells. After being grown at 37°C with 5% CO2, the cells were rinsed with PBS and cultured in fresh medium overnight. The next day, cells were treated with hemin for immunofluorescence and RNA sequencing. The G4 staining profile used in our study is available for download from figshare (https://doi.org/10.6084/m9.figshare.21608127.v1 ).
Adeno-associated viral (AAV) production and transduction
The shRNA targeting murine Hmox1 was constructed using the AAV-U6sgRNA (SapI)-hSyn-GFP-KASH-bGH vector (Addgene #60958). The sequence of shHmox1 was 5′-ACC GAG CCA CAC AGC ACT ATG TAA ATT CAA GAG ATT TAC ATA GTG CTG TGT GGC TTT TTT TG-3′. For the production of recombinant AAV, HEK293T cells were seeded in 150-mm plates and co-transfected with pAAV-shHmox1, pAAV2/8-RC, and pHelper (1:1:1 molar ratio) using PEI MAX 40K (PolySciences). Seventy-two hours post-transfection, the cell culture supernatants were harvested, filtered with a 0.22-μm filter, and were further concentrated with Amicon Ultra-15 centrifugal filters (Millipore). The titer of recombinant AAV was determined by quantitative PCR as previously published . To detect AAV knockdown efficiency, recombinant AAV viruses were used to transduce to the MEF cells. MEF cells were seeded in a 6-well plate and transduced by AAV with a multiplicity of infection (MOI) of 100,000 vgs per cell. Twenty-four hours post-transduction, cells were washed three times with PBS and cultured for another 48 h before collection. The cells were then analyzed by quantitative RT-PCR and western blot with an anti-HMOX1 antibody.
Knockdown of Hmox1 by AAV in vivo
Eight-week-old C57BL/6 mice were injected with the recombinant AAV (5×1011 viral genome AAV8-shNC and AAV8-shHmox1 diluted to 200 μL with PBS) through tail vein injection. Serum alanine aminotransferase (ALT) was monitored with the ALT assay kit (Nanjing Jiancheng Bioengineering Institute, C009) following the manufacturer’s instructions. Mice exhibiting increased serum ALT levels were euthanized, and their liver tissues were dissected for Hmox1 RT-PCR analysis or hemin quantification. Collected mouse liver tissues were fixed with 4% neutral buffered formalin and embedded in paraffin. After deparaffinization, the sections were sequentially stained with the alum hematoxylin and eosin. Following dehydration and clearing, the sections were mounted in neutral balsam and imaged with Vectra 3 automated quantitative pathology imaging system (Akoya Biosciences). All animal experiments were conducted according to the guidelines of the ethics committee of the animal facility, Wuhan University. All mice were specified as pathogen-free and were housed under controlled temperature and light conditions following the animal care guidelines.
Quantification and statistical analysis
Data are presented as Mean ± SD. The peak or gene size (N) in the heatmaps and scatter plots indicates the number of peaks or genes included. The sample sizes (n) in the figure legends indicate the number of replicates in each experiment. Statistical analyses in Fig. 3B–D, F–H, Figs. 4G–I, S3E, S5A, S5C, and S5D were performed by unpaired Student’s t test and the p values were denoted in each figure. The nested t-test was used for statistical analysis in Fig. 7B. Kolmogorov-Smirnov test (K-S test) was used for statistical test in Figures S3H and S3J.