Agents and antibodies

Reagents in this study were obtained as following. MS417 (#HY-111,139) from MedChemExpress (New Jersey, USA); DMEM/F12 (#ZQ600) from Zhong Qiao Xin Zhou Biotechnology (Shanghai, China); Oil Red O (ORO) staining kit (#0843) from ScienCell (California, USA); BODIPY 493/503 (#D2191), EGS (#21,565), GeneRuler 50 bp (#SM0373), 16% Formaldehyde Solution (#28,906), ChIP-Grade Protein A/G Magnetic Beads (#26,162) and RNase A (#EN0531) from Thermo Fisher Scientific (Boston, USA); Palmitic acid (#P9767) from Sigma-Aldrich (Missouri, USA); Albumin BovineV (fatty acid-free; #A8850) and Nigericin sodium salt (#IN1880) from Solarbio (Beijing, China); 4’,6-diamidino-2-phenylindole (DAPI; #C1006), Protein inhibitor cocktail (#P1005), Lipo8000 transfection reagent (#C0533), DAB Horseradish Peroxidase Color Development Kit (#P0203), RIPA lysis buffer (#P0013B), and Enhanced BCA protein assay kit (#P0010) from Beyotime (Shanghai, China); RNAiso Plus from Takara (#108-95-2, Tokyo, Japan); GoScript™ Reverse Transcription Mix and Random Primers (#A2800) and Eastep™ qPCR Master Mix (#LS2068) from Promega (Wisconsin, USA); Viability dye 506 (#62210-00) from Biogems (California, USA); 0.5 M EDTA (#7011), and 10X Glycine (#7005S) from Cell Signaling Technology (Boston, USA); 6X Gel loading dye (#B7024S), Micrococcal Nuclease (#M0247S), NEBNext Ultra™ II DNA Library Prep Kit for Illumina (#E7645S) and Multiplex Oligos for Illumina (#E7600s) from New England Biolabs (Ipswich, USA); AMPure XP (#A63881) from Beckman Coulter (California, USA). Penicillin, Streptomycin, and trypsin-ethylenediaminetetraacetic acid from Genom Biotechnology (Shanghai, China). All antibodies in this study were listed in Table S1.

Isolation and culture of primary hepatocytes

From wild-type C57BL6/J mice, primary hepatocytes were isolated using the collagenase perfusion approach that has been previously described [8]. In summary, newly extracted hepatocytes were seeded into collagen I-coated petri plates and were cultivated in RPMI medium 1640 supplemented with 1.0 g/L glucose, 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/mL streptomycin at 37 ℃ in a humidified environment. Six hours after plating, the medium was changed, and the cells received the appropriate treatment.

Cell treatment and transfection

Mouse hepatocyte cell line AML12 obtained from Zhong Qiao Xin Zhou Biotechnology (Shanghai, China) were cultivated in DMEM/F12 containing 10% FBS, 50 U/mL penicillin, and 50 µg/mL streptomycin. The cells were incubated in a humidified environment composed of 95% air and 5% CO2 at 37 ℃. AML12 or primary hepatocytes were seeded in 6-well plates in serum-containing media and were cultured to 70-80% confluence followed by serum-starved for 24 h. Subsequently, AML12 or primary hepatocytes were induced by 500 µM PA as an in vitro model of hepatocellular lipotoxicity [25,26,27]. Briefly, PA was firstly conjugated to bovine serum albumin (BSA) [27]; then was added in the medium to induce oxidative stress and lipoapoptosis in AML12 or primary hepatocytes.

MS417, a selective inhibitor of the BET-specific protein BRD4, was chosen to apply in AML12 or primary hepatocytes. Therefore, after the cells reached 70-80% confluence and were starved for 24 h, AML12 or primary hepatocytes were challenged with 500 µM PA or BSA (control) in serum-containing media for 24 h, with or without MS417 (50 nM).

AML12 or primary hepatocytes were transfected with siRNA for BRD4 to transiently knock down BRD4. Non-targeted scrambled control siRNA (NC siRNA) and 3 groups of BRD4 siRNA (#1, #2, and #3) were transfected into AML12 cells using Lipo8000 transfection reagent according to the manufacturer’s instruction. The BRD4 siRNA and NC siRNA were provided by Genepharma (Shanghai, China), and the produced oligo sequences are displayed in Table S2. BRD4 knockdown efficiency was verified by qPCR and Western blotting, respectively (Fig. S1). With the highest knockdown efficiency, siBRD4 (#1) was finally chosen to transfect AML12 hepatocytes. 48 h after transfection, cells were starved and then incubated with BSA (control) or PA accompanied by MS417 or not for another 24 h.

Human liver specimens

Human liver specimens were obtained from patients with MASLD/MASH who underwent liver biopsy or surgical liver resection, and healthy liver tissues were obtained from the unaffected part of the liver from patients undergoing surgery for hepatic hemangioma or trauma repair. All liver samples came from Shanghai Public Health Clinical Center. Subjects with other chronic liver diseases were excluded, including viral hepatitis, cholestatic liver disease, excessive alcohol consumption, Wilson disease, hemochromatosis, drug-induced liver disease, and α-1-antitrypsin deficiency. All subjects gave written informed consent according to the Declaration of Helsinki, and the protocol, approved by ethical committees from Shanghai Public Health Clinical Center (#2022-S104-01).

MASH liver samples

Mice liver samples including MASH and the control group were obtained from our recent cohort study [28]. In brief, male C57BL/6J mice aged eight weeks (Shanghai Slack Laboratory Animal Co., Ltd., Shanghai, China) were maintained in an SPF facility with an environmental control system. Mice had unrestricted access to food and liquids with a 12-hour light-dark cycle. Mice were fed either a normal chow diet (NCD) or a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD; Research Diets, USA) for 8 weeks [28].

Histopathology and immunohistochemistry

For histopathological assessment, paraffin-embedded formalin-fixed liver sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome, or Sirius red according to standard protocols [28]. Liver histology was blinded reviewed by two independent expert pathologists and MASH was defined by the NAFLD activity score (NAS) as follow [27]. Briefly, steatosis (0–3), lobular inflammation (0–3), and hepatocellular ballooning (0–2); and NAFLD activity score (NAS) was expressed as the sum of each scoring. Additionally, liver fibrosis and lipid droplet areas were also assessed blindly using ImageJ software based on Sirius red and H&E-stained sections, respectively.

Immunohistochemical (IHC) staining was carried out as our previously described [28, 29]. Briefly, 5 μm-thick sections were de-paraffinized, rehydrated, and subjected to heat-induced epitope retrieval. Then, slides were blocked and incubated overnight at 4 ℃ in the following primary antibodies (dilution 1:100): anti-BRD4, anti-VADC1, anti-NLRP3, and anti-GSDMD. The list of the antibodies is provided in Table S1. Following washing in PBS, sections were subsequently incubated with HRP-conjugated secondary antibodies (dilution 1:100) and developed using DAB Horseradish Peroxidase Color Development Kit according to the manufacturer’s protocol. Finally, the positive areas were examined under a light microscope (BX43, Olympus). Immunostaining signaling was quantified at a predetermined threshold using free NIH ImageJ.

Immunofluorescence

The hepatocytes were dehydrated at 37 ℃ for 30 min and fixed with 4% paraformaldehyde for 30 min followed by permeabilization using 0.2% TritonX-100 in PBS. Then nonspecific binding was blocked with 5% BSA for 1 h at RT, followed by incubation with primary antibodies for BRD4 (dilution 1:100) and HNF4 (dilution 1:100) at 4 ℃ overnight. After washing in PBS, the cells were incubated with secondary conjugated antibodies for 1 h at 37 ℃. DAPI was used for nuclear staining. The slides were finally imaged by a confocal microscope (FV3000, Olympus).

Oil red O and BODIPY staining

Lipid accumulation was analyzed using an Oil Red O (ORO) staining kit as described in the manufacturer’s protocol. AML12 cells or primary hepatocytes were washed with PBS, and fixed with 4% paraformaldehyde for 15 min. Then, cells were stained with freshly diluted 0.6% ORO solution preheated to 65 ℃ for 1–5 min. Cell nuclei were stained by hematoxylin for 10 s. The stained lipid droplets were observed under a light microscope (BX43, Olympus).

For BODIPY staining, the cells were treated and fixed the same with ORO staining. Then the fixed cells were stained with BODIPY 493/503 dye (dilution 1:1000) at RT for 10 min and protected from light, and the nuclei were stained with DAPI. The stained lipid droplets were observed under a confocal microscope (FV3000, Olympus). The areas of the lipid droplets in different groups were analyzed using NIH Image J 1.49 software.

Western blot assay

Western blot analysis was performed according to standard protocols as previously described [28, 29]. Cells and liver tissues lysed in RIPA buffer were centrifuged at 4 ℃, and protein concentrations were assessed using the Enhanced BCA protein assay kit. Protein (10–20 µg) was resolved by 12.5% SDS-PAGE gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked and incubated overnight at 4 ℃ with primary antibodies (dilution 1:1000) against BRD4, β-Actin, NLRP3, total and cleaved GSDMD, pro- and cleaved-Caspase-1, IL-1β, apoptosis-associated speck-like protein (ASC), VDAC1, and H3K27ac. Followed by washing in TBST, the membranes were incubated with a 1:5000 dilution of peroxidase-conjugated secondary antibodies under RT for 1 h. β-actin was used as loading control. Bands were visualized with ECL™ Western Blotting Detection Reagents (EpiZyme, Shanghai, China), and the optical density of the bands was determined using the NIH ImageJ software.

VDAC cross-linking assay

Briefly, AML12 hepatocytes treated as indicated were washed twice with PBS, and incubated with Ethylene Glycol-bis (Succinimidylsuccinate, EGS), a chemical cross-linking reagent wich diluted in PBS, at 30 ℃. Then the lysed protein samples from AML12 cells were subjected to SDS-PAGE and immunoblotting analyzed with anti-VDAC1 antibody. After time gradient and concentration gradient experiments (Fig. S2A, B), we finally selected 600 µM EGS for 30 min as the optimal incubation conditions [30].

RNA isolation and real-time quantitative PCR

Total RNA was extracted using RNAiso Plus according to the manufacturer’s instructions [28]. For qPCR, total RNA was reverse transcribed to cDNA using Promega GoScript™ Reverse Transcription Mix and Random Primers. Relative quantitative gene expression levels were measured by quantitative PCR using Eastep™ qPCR Master Mix on QuantStudio 5 System (Applied Biosystems). β-actin was used as an internal control, and the relative expression of target genes was calculated using the 2−ΔΔCT method. All primers used are listed in Table S3.

Flow cytometry analysis

Cell viability was measured using fixable Viability dye 506. Briefly, AML12 cells grown in six-well plates were treated as indicated and harvested. Then, cells were resuspended in PBS at a concentration of 1–10 × 106/mL. Add 1 µL of Viability Dye per 1 mL of cells, then vortex immediately, after that cells were incubated for 15 min at RT in the dark. Cells were washed three times with PBS buffer before testing, and flow cytometry was employed to detect cell viability. The Viability Dye 506 can be excited by the violet (405 nm) laser and has a peak emission of 506 nm detectable by the 510/50 bandpass filter. The dead cells were identified by positive signals. Data were analyzed using the FlowJo software (V10.8.0).

Chromatin immunoprecipitation (ChIP)

For ChIP, AML12 cells were seeded in 15 cm culture dishes and were challenged with PA with or without the co-culturation of MS417. For ChIP, protease inhibitor cocktail (#P1005, Beyotime) was used in all buffers. Cells were PBS washed and fixed for 10 min at RT with shaking in fresh methanol-free 1% formaldehyde, then quenched by 0.125 M glycine. Cells were lysed in lysis buffer LB1 (50mM Hepes-KOH, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100) and LB2 (10mM Tris-HCl, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) and incubated at 4 ℃ for 10 min, respectively. Then cells were resuspended in LB3 (10mM Tris-HCl, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, 0.2% SDS) and were sonicated to shear DNA to 250–500 bp. The sonicated samples were centrifuged at 10,000 rcf, 10 min at 4 ℃, then the supernatant was taken and the DNA concentration was detected. The samples containing 5–10 µg of DNA were taken and incubated with anti-BRD4, anti-H3K27ac antibody (1 µl per 5 µg samples), or BSA for input DNA overnight at 4 ℃.

The next day, ChIP-Grade Protein A/G Magnetic Beads (20 µl per 5 µg samples) were added to each ChIP sample at RT and incubated at 4 ℃ for 2 h with rotation to collect chromatin/antibody. Beads were collected with a magnet and washed in RIPA buffer (1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, TE), RIPA buffer with 5 M NaCl, LiCl buffer (0.25 M LiCl, 0.5% NP-40, 0.1% Na-deoxycholate, TE), TE with 0.2% Triton X-100 and TE, succeedingly. After proteinase K digestion and reverse crosslinking, the precipitated DNA and input DNA were purified using a PCR purification kit and subjected to DNA library construction.

For ChIP-sequence (ChIP-seq) library preparation, samples were performed using NEBNext Ultra™ II DNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina according to the manufacturer’s recommendations. DNA libraries were sequenced on an NOVAseq600 instrument, with 150-bp paired-end sequencing [31].

ChIP-seq analysis

Raw reads were filtered using cutadapt (version 1.15). Clean reads were aligned to the mouse reference genome GRCm38 (mm10) using Bowtie2 aligner (version 2.3.3.1) with default parameters. MACS2 was used to call the broad peaks with -q 0.05 [31]. Differentially bound peaks were identified with DiffBind (version 3.13) [32]. The peaks were annotated with the annotatedPeaks.pl command from HOMER (version 4.9) to associate peaks to their nearest genes. BigWig files were generated using the bamCoverage command from deeptools (version 3.5.0) with -smoothLength 50 –binSize 5 and –scaleFactor, and markdup.bam files were as input for bamCoverage [32]. The scaleFactors were normalization factors calculated from DiffBind based on the number of all mapped reads that fall into the called peak regions. ChIP-seq heatmaps and profile plots were generated with computeMatrix followed by the plotHeatmap command from deeptools (version 3.5.0). Merged BigWig files of PA/Vehicle-anti H3K27ac, PA/MS417-anti H3K27ac, PA/Vehicle-input, and PA/MS417-input were generated from two biological replicates with the bigWigMerge command followed by the bedGraphToBigWig command from UCSC [32]. The gene set enrichment analysis for differentially bound genes was conducted using KEGG pathway analysis.

Statistical analysis

All data are presented as the mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 9.0 (La Jolla, CA, USA). Statistically significant differences between two independent groups were made by unpaired two-tailed Student’s t-test. Comparisons among multiple groups were performed by one-way ANOVA with Tukey’s post hoc honest significant difference test or two-way ANOVA followed by Tukey’s or Sidak’s multiple comparisons test. Clinical characteristics and NAS scores were corrected using Spearman’s rank correlation test. For all comparisons, significance is indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns indicates not significant.