Human samples

A cohort of 31 female patients aged 23 to 40 years who underwent laparoscopy for ovarian endometrioma (OE) were recruited from Nanfang Hospital, Southern Medical University, and from the First Affiliated Hospital of Hainan Medical University. Additionally, a control group of 23 women without endometriosis who were undergoing laparoscopic or hysteroscopy for other benign gynecological diseases, such as tubal disease or uterine myomas, was established. All participants had regular menstrual cycles, with their menstrual history duly confirmed. The diagnosis of ovarian endometrioma was substantiated through meticulous histological examination. Preceding surgical interventions, patients were required to refrain from hormonal treatments and intrauterine contraception for a minimum of 6 months. OE samples comprised tissues from ovarian endometriotic cysts (n = 31) and their eutopic proliferative phase endometrium (n = 31), while the control group comprised tissues from the eutopic proliferative phase endometrium (n = 23). All participants were in the proliferative phase of their menstrual cycles. Samples from these participants were divided into two portions. One portion was immediately collected to isolate primary human EcESCs, eutopic endometrial stromal cells (EuESCs), and normal endometrial stromal cells (NESCs) for a series of in vitro experiments, including RNA isolation, protein extraction, lentivirus infection, collagen gel contract, and migration assays. The other portion was fixed for immunofluorescence and immunohistochemistry experiments. All participants signed informed consent for collection of their endometrial and endometriotic tissues. The basic information of all participants is shown in Additional file 1: Tables S1, S2.

Analysis of published single cell RNA-seq data

The single-cell transcriptomic data used in this study were retrieved from the NCBI Gene Expression Omnibus with the GEO series number GSE213216 [20]. To ensure data quality, low-quality cells were excluded based on the following criteria: (1) < 200 genes; or (2) > 20% unique molecular identifiers (UMIs) derived from the mitochondrial genome. Doublets were identified and removed using the R package, DoubletFinder (v.2.0.3), under the default settings. The batch effect across different samples was removed using the R package, harmony (v.0.1.1). The standard procedures of filtering, variable gene selection, dimensionality reduction and clustering were performed using the single cell RNA seq analysis R package Seurat (v.4.3.0). Mesenchymal cells were defined by the expression of cell type-associated genes: DCN, COL11A2, FAP, PDGFRA, COL11A1, COL1A1, and PDGFRB. Mesenchymal cell types were further sub-clustered to further decode the mesenchymal landscape in endometriosis. Scaling, principal component analysis, and clustering were performed as described above. Subsequently, the subclusters were annotated based on reported cell-specific marker genes [20]. Differential expression of the various clusters was also analyzed using Seurat, employing the Wilcoxon rank sum test.

Fig. 1

KAT14 was upregulated in human ovarian endometrioma lesions and primary human EcESCs. (A) Representative immunohistochemical staining for KAT14 in human normal endometrium tissue (n = 8), eutopic endometrium (n = 9), and ectopic lesions (n = 9) from human ovarian endometrioma. Scale bar: 100 μm (upper panel), scale bar: 50 μm (lower panel). (B) Double labeling immunofluorescence analysis showing the expression and distribution of KAT14 and α-SMA within normal endometrial tissues (n = 8), eutopic endometrium tissues (n = 9), and ectopic lesions (n = 9) from patients with ovarian endometrioma. Scale bar: 50 μm. (C) Single-cell differential expression analyses of histone acetylation genes in endometriomas, endometriosis, and eutopic endometrium. Each point (hollow circle) represents a single histone acetylation gene. The X-axis displays the average logarithm of fold change (logFC) values for genes measured in activated fibroblasts derived from endometrioma and endometriosis compared to those from the eutopic endometrium. The Y-axis represents the average logFC values for genes measured in all activated fibroblasts from endometrioma and endometriosis in relation to other cell populations from the same conditions. (D) qRT-PCR analysis of KAT14, FN1, COL1A1, and ACTA2 transcripts in primary EcESCs (n = 6) compared to NESCs (n = 6) and EuESCs (n = 6). Relative quantification of gene expression was calculated using the 2−∆∆Ct method and normalized to GAPDH as the internal control. (E) Western blot was used to measure the protein level of KAT14, Fibronectin1, Collagen I, and α-SMA in primary EcESCs (n = 11) compared to NESCs (n = 8) and EuESCs (n = 11). Data are representative of three or more independent experimental replicates. For all panels, data are presented as the mean ± SD. P-values were determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: Not significant. EMs: endometriomas, EcESCs: ectopic endometrial stromal cells, EuESCs: eutopic endometrial stromal cells, NESCs: normal endometrial stromal cells

Fig. 2

KAT14 overexpression exacerbated TGF-β1-induced fibrogenesis in immortalized HESCs. (A) Bar graph showing the enrichment analysis of upregulated genes after KAT14 overexpression in terms of GO molecular function. (B) Gene set enrichment analysis (GSEA) of DEGs induced by KAT14 overexpression compared to the negative control showing significant enrichment in gene sets associated with extracellular matrix structural constituents and ECM component pathways. Normalized enrichment score (NES), false discovery rate (FDR), and P-values are shown. (C) Heatmap profile of DEGs associated with fibroblast activation and ECM remodeling in KAT14-overexpressed cells compared to control cells based on RNA-Seq (n = 3). (D) qRT-PCR analysis of the relative mRNA expression of FN1, COL1A1, and ACTA2 in HESCs infected with the indicated lentiviruses harboring KAT14 expression vector (pCDH-KAT14) or empty vector control (pCDH-Ctrl) treated with TGF-β1 for 24 h. Relative quantification of gene expression was calculated using the 2−∆∆Ct method and normalized to GAPDH as the internal control. (E) Western blots measuring the protein level of fibronectin, collagen I, and α-SMA in HESCs infected with pCDH-KAT14 or pCDH-Ctrl lentiviruses under TGF-β1 stimulation for 48 h. (F) Immunofluorescence staining showing the expression of KAT14, α-SMA, and the ECM molecules fibronectin and collagen I in HESCs infected with pCDH-KAT14 or pCDH-Ctrl lentiviruses under TGF-β1 stimulation for 48 h. Scale bar: 50 μm. (G) Collagen gel contractility assay showing the cell contraction capacity of HESCs infected with pCDH-KAT14 or pCDH-Ctrl lentiviruses under TGF-β1 stimulation for 24 h. The degree of collagen gel contraction was determined as the difference between the diameters of the well and the released gels. (H) Wound healing assay showing the migration ability of HESCs infected with lentiviral vector containing pCDH-KAT14 or pCDH-Ctrl treated with TGF-β1 for 0, 18, and 36 h. Wound healing was assessed by calculating the area in µm2 between the lesion edges. Scale bar: 200 μm. TGF-β1 was used at a concentration of 12 ng/ml. Data are representative of three or more independent experimental replicates. For all panels, data are presented as the mean ± SD. P-values were determined by Student’s t-test in panels (D, E, G, H). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. HESC: human endometrial stromal cell, GO: gene ontology, DEGs: differentially expressed genes, Ctrl: control

Fig. 3

Knockdown of KAT14 attenuated TGF-β1-induced fibrogenesis in primary human EcESCs. (A) Western blot showing the protein levels of fibronectin, collagen 1, and α-SMA in primary EcESCs infected with shRNA-KAT14 or shRNA-Ctrl lentiviruses treated with TGF-β1 or vehicle for 0, 48, and 72 h. **P < 0.01, ***P < 0.001, and ****P < 0.0001 shRNA-KAT14 group versus shRNA-Ctrl group; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 shRNA-Ctrl group at 48–72 h versus shRNA-Ctrl at 0 h. (B) qRT-PCR analysis of the relative mRNA expression of FN1, COL1A1, and ACTA2 in primary EcESCs infected with shRNA-KAT14 or shRNA-Ctrl lentiviruses treated with TGF-β1 or vehicle for 24 h. Relative quantification of gene expression was calculated using the 2−∆∆Ct method and normalized to GAPDH as the internal control. (C) Western blots were used to measure the protein level of fibronectin, collagen I, and α-SMA in primary EcESCs infected with shRNA-KAT14 or shRNA-Ctrl lentiviruses stimulated by TGF-β1 or vehicle for 24 h. (D) Immunofluorescence staining showing the expression of KAT14, HESC activation marker, α-SMA, and the ECM molecules fibronectin and collagen I in primary EcESCs infected with shRNA-KAT14 or shRNA-Ctrl lentiviruses stimulated by TGF-β1 or vehicle for 48 h. Scale bar: 50 μm. (E) Collagen gel contractility assay showing the cell contraction capacity of primary EcESCs transfected with shRNA-KAT14 or shRNA-Ctrl lentiviruses stimulated by TGF-β1 or vehicle for 24 h. The degree of collagen gel contraction was determined as the difference between the diameters of the well and the released gels. (F) Wound healing assay showing the migration ability of primary EcESCs infected with shRNA-KAT14 or shRNA-Ctrl lentiviruses stimulated by TGF-β1 or vehicle for 0, 24, and 48 h. Wound healing was assessed by calculating the area in µm2 between the lesion edges. Scale bar: 200 μm. The concentration of TGF-β1 used for panels (A–F) was 12 ng/ml. Data are representative of three or more independent experimental replicates. For all panels, data are presented as the mean ± SD. P-values were determined by Student’s t-test in panel (A) and by one-way ANOVA in panels (A–C, E, F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. EcESCs: ectopic endometrial stromal cells, Ctrl: control

Animal study

Thirty 6-week-old female C57BL/6 mice (RRID: IMSR_JAX:000664) were purchased from ZHBY Biotech Co., Ltd (Nanchang, China). Adult female mice (6 weeks) underwent a 1-week quarantine period and were subsequently employed to establish experimental models under specific pathogen-free conditions. An established mouse-mouse intraperitoneal transplantation model, as outlined in prior studies [21] with modifications, was used to generate the endometriosis (EMs) model. To eliminate potential variation, the endometrial tissue fragments from two donor mice were mixed and then divided into four parts, each stitched to two mice drawn from one of the two experimental groups on day 0, as per the experimental design. The sham group was established using the same steps as the endometriosis surgeries, excluding the suturing of any tissue to the abdominal wall. Eight mice were randomly selected as donors for this experiment, and EM mouse models were successfully established in 14 recipient mice. For KAT14 knockdown assays, EM mice were randomized into two groups (n = 6 per group): (1) EM mice treated with AAV9 carrying shRNA for mouse KAT14 (AAV9-shKAT14); and (2) EM mice treated with the negative control (AAV9-shCtrl). Both groups received local injections of AAV9 carrying shKAT14 or its negative control shCtrl on day 14 after endometrial tissue implantation. Mice were euthanized on day 28, and uterine and ectopic lesions were collected for subsequent studies. The detailed animal experiment process is shown in Fig. 4A.

Fig. 4

KAT14 knockdown ameliorates endometriosis-associated fibrosis in vivo. (A) Schematic diagram of the in vivo experiments. (B) qRT-PCR analysis of Fn1, Col1a1, and Acta2 expression in eutopic endometrium tissues from sham control, ectopic endometrial-like lesions, and eutopic endometrium tissues from endometriosis mice locally injected with AAV9-shKAT14 or its negative control (AAV9-shCtrl). Relative quantification of gene expression was calculated using the 2−∆∆Ct method and normalized to Gapdh as the internal control; n = 6 mice per group. (C) Western blotting analysis of fibronectin, collagen I, and α-SMA expression in eutopic endometrium tissues from sham control, ectopic endometrial-like lesions, and eutopic endometrium tissues from endometriosis mice injected with AAV9-shCtrl or AAV9-shKAT14; n = 6 mice per group. (D) Representative images of collagen I and α-SMA immunohistochemistry and Masson’s staining in eutopic endometrium tissues from sham control, ectopic endometrial-like lesions, and eutopic endometrium tissues from AAV9-shCtrl injected or AAV9-shKAT14 injected endometriosis mice; n = 6 mice per group, Scale bars: 50 μm. (E) Representative triple immunofluorescence staining images of collagen I, α-SMA, and fibronectin in eutopic endometrium tissues from sham control, eutopic endometrium tissues from sham control, ectopic endometrial-like lesions, and eutopic endometrium tissues from AAV9-shCtrl injected or AAV9-shKAT14 injected endometriosis mice; n = 6 mice per group, Scale bars: 50 μm. Data are shown as the mean ± SD. P-values were determined by one-way ANOVA. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: Not significant, AAV: adeno-associated virus

Cell lines and cell culture

The immortalized human endometrial stromal cell line, generously provided by Prof. Haibin Wang (Xiamen University), underwent cell immunofluorescence staining to determine the purity of the human endometrial stromal cells (HESCs) using pan-cytokeratin (#26411-1-AP, Proteintech, RRID: AB_2880505), CD10 (#23898-1-AP, Proteintech, RRID: AB_2879354), and vimentin (#60330-1-Ig, Proteintech, RRID: AB_2881439) (Additional file 1: Fig. S7A), as previously described [22]. The HESCs were cultured according to established protocols [23] involving a maintenance medium of DMEM/F-12 containing 10% (v/v) charcoal-stripped fetal bovine serum (CS-FBS, #04-201-1 A, BIOLOGICAL INDUSTRIES), 3.1 g/L glucose (#D823520, Macklin), and 1 mM sodium pyruvate (#S817535, Macklin) supplemented with 1% penicillin–streptomycin, 1.5 g/L sodium bicarbonate (#S818079, Macklin), 500 ng/ml puromycin (#P8230, Solarbio), and 1% insulin–transferrin–selenium (#51,500,056, Thermo Fisher) at 37 °C and 5% CO2. Wild-type HESCs or established stable KAT14-KO HESCs were stimulated with 12 ng/ml TGF-β1 (#100 − 21, PEPROTECH) or vehicle (PBS) and harvested for subsequent experiments.

Primary endometrial stromal cells (ESCs) were isolated and cultured as previously described [22]. In brief, the proliferative phase endometrial and endometriotic tissues were washed with sterile D-Hanks Balanced Salt Solution (HBSS, #H1045, Solarbio) and 1% penicillin–streptomycin (#15,140,122, Gibco) and then carefully dissected and minced into small pieces, approximately 1 mm3 in size. The minced tissues were incubated in HBSS containing type IV collagenase (4%) (#17,104,019, Gibco) in a shaking incubator for 90 min at 37 °C. The undigested tissue was removed by centrifugation at 200 g for 3 min. The cell suspension was passed through a graduated series of nylon mesh cell strainers (70 μm and 40 μm) (#352,350, BD; #352,340, BD) and then washed three times by hypotonic lysis (#R1010, Solarbio) to remove the erythrocytes. Finally, the ESC pellets were resuspended in a maintenance medium composed of DMEM/F-12 supplemented with 10% fetal bovine serum (FBS; #10270-106, Gibco) and 1% penicillin–streptomycin, plated on a 60-mm cell culture dish (#430,166, Corning), and incubated at 37 °C in 95% air/5% CO2. Forty-eight hours after cells seeding, the original medium was replaced and the unattached epithelial cells were removed by rinsing with phosphate buffered saline (PBS) (#C10010500BT, Gibco). The ESCs were passaged and used before the fourth passage for experiments. Next, cell immunofluorescence staining was conducted to determine the purity of the isolated ESCs using antibodies for human pan-cytokeratin (#26411-1-AP, Proteintech, RRID: AB_2880505), CD10 (#23898-1-AP, Proteintech, RRID: AB_2879354), and Vimentin (#60330-1-Ig, Proteintech, RRID: AB_2881439 (Additional file 1: Fig. S6A), as previously described [22]. In some experiments, primary ESCs were cultured with either TGF-β1 or the SRF inhibitor CCG-1423 (#SML0987, Sigma) for 24–48 h, with CCG-1423 dissolved in DMSO (#D2650, Sigma), while the same volume of DMSO was added to the controls.

Human embryonic kidney 293T cells (HEK 293T) (RRID: CVCL_0063) were purchased from IGE Biotechnology Co. Ltd. (Guangzhou, China) and cultured at 37 °C and 5% CO2, with regular passaging. The HEK 293T cell line underwent short tandem repeat profiling for identification and was confirmed to be free of mycoplasma contamination before the experiments.

RNA sequencing (RNA-seq) and data analysis

HESCs were cultured in DMEM/F-12 medium and transfected with pCDH-KAT14 or pCDH-control lentiviruses. After treatment, total RNA was extracted using TRIzol reagent (#15596-018, Invitrogen) following the standard protocol. Then, the RNA quality and quantity were assessed using a NanoDrop One/OneC Microvolume UV-Vis spectrophotometer (Thermo Fisher Scientific) and Qubit 2.0 Fluorometer (Invitrogen), the integrity of RNA was confirmed using an Agilent 4200 TapeStation System. The cDNA libraries were generated by HaploX Biotechnology (Jiangxi, China). Specifically, the transcriptome library for sequencing was generated using the NEBNext Ultra™ II mRNA Library Prep Kit for Illumina kit (#E7770L, NEB) according to the manufacturer’s specifications. Furthermore, the library fragments were qualified/quantified using TapeStation4200 (Agilent). The RNA-seq libraries were sequenced on the Illumina PE150 system. Raw data in fastq format were processed with fastp. Clean data were obtained after quality control, adapter trimming, quality filtering, and per-read quality cutting. All of the downstream analyses were based on the clean data with high quality. Then, paired-end clean reads were aligned to the reference genome using HISAT2 software v2.1.0 [24]. Differential gene expression (|log2(FoldChange)| > 1 and adjusted P-value < 0.05) analysis of two groups was performed using the DESeq R package (1.18.1) [25]. Gene Ontology (GO) enrichment analysis and visualization of differentially expressed genes was implemented by the ClusterProfiler R package [26]. Gene set enrichment analysis (GSEA) was performed using GSEA software v4.1.0 [27].

Lentiviral vectors and stable cell line construction

The total RNA from immortalized human HESCs was isolated using TRIzol following the manufacturer’s protocol. The full length coding sequence of human wild-type KAT14 (NM_001392973.1) cDNA was synthesized by reverse transcription-PCR (RT-PCR) using the HiScript II 1st Strand cDNA Synthesis Kit (#R211-01, Vazyme). To generate plasmids for mammalian expression of KAT14, KAT14 cDNA fused with an HA tag at the N-terminal region was subcloned into the pCDH-CMV-MCS-EF1-puro plasmid (IGE Biotechnology Co. Ltd., Guangzhou, China). All constructs were confirmed by direct sequencing. The shRNA (short hairpin RNA) targeting KAT14 was designed from the website https://www.sigmaaldrich.cn/CN/zh. After annealing, the double-stranded oligonucleotides were subcloned into the EcoRI and AgeI restriction sites of pLKO.1 vectors (#8453, Addgene, RRID: Addgene_8453).

All lentiviruses used in this study (viral particles containing plasmids that express KAT14 or the shRNA-KAT14) were generated in HEK 293T cells via polyethyleneimine linear (#40816ES02, YEASEN) transfection, as previously described [28]. For lentivirus infection, target cells were seeded in a 24-well plate (#3513, Corning) (4 × 104 cells per well), cultured for 24 h, and then incubated with viral particles. To establish stable KAT14-knockdown (KD) cell lines, primary human EcESCs were transfected with shRNA-KAT14 or shRNA-control lentiviruses. For the KAT14 rescue experiment, KAT14 overexpression (pCDH-KAT14) lentiviruses and the empty vector were transfected into the previously established stable KAT14-knockout (KO) immortalized HESCs or the established stable KAT14-KD EcESCs. Cells infected with lentivirus were selected with Puromycin (#P8230, Solarbio) (2.5 µg/ml for immortalized HESCs, 4 µg/ml for EcESCs) to eliminate non-infected cells 72 h after infection, yielding stably transfected cell lines for subsequent experiments. Detailed information on these shRNA target sequences is provided in Additional file 1: Table S3.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 KAT14 knockout

For genome editing in immortalized HESCs, single guide RNA (sgRNA) targeting KAT14 was designed using an optimized CRISPR online tool (http://crispr.mit.edu/), as previously described [29]. Specific KAT14 sgRNA oligos were annealed and cloned into lentiCRISPR v2 (#52,961, Addgene, RRID: Addgene_52961), encoding the Cas9 protein. Lentivirus packaging was performed as described above. Immortalized HESCs were seeded in a 24-well plate (4 × 104 cells per well) and incubated with virus supernatant. Seventy-two hours after infection, immortalized HESCs were screened with 2.5 µg/ml puromycin for 1 week to select stably transfected cell lines for further experiments. The puromycin-selected cells were diluted into 96-well plates (#3599, Corning) at densities of 15–20 cells/ml to obtain individual cells for further single cell clone establishment. For each clonal population, 50 to 100 cells were harvested to isolate genomic DNA 12–14 days after cell seeding and subjected to direct PCR to amplify fragments containing the editing site. The knockout efficiency of cells was validated by sequencing the PCR amplification products and western blotting. Detailed information of the sgRNA target sequences are shown in Additional file 1: Table S3.

Small interfering RNA (siRNA) transfection

The established stable KAT14-KD primary human EcESCs were seeded onto 100-mm cell culture dishes (#430,167, Corning) for chromatin immunoprecipitation, 6-well plates (#3516, Corning) for western blot assay, or 24-well plates for luciferase assay and gel contraction assays. The cells were transfected with siRNA-SRF and the nonsense mutation negative control (siRNA-ctrl) (IGE Biotechnology Co. Ltd., Guangzhou, China) at approximately 60% confluence. siRNA transfections were performed in serum-free OptiMEM (#31,985,062, Gibco) using Lipofectamine 3000 (#L3000008, Invitrogen) according to the manufacturer’s protocol. After 36 h of incubation, the culture media was refreshed and cells were stimulated with 12 ng/ml TGF-β1 or PBS (#C10010500BT, Gibco) for 24 h before conducting subsequent experiments. Detailed information on the siRNA target sequences are shown in Additional file 1: Table S3.

RNA isolation, cDNA synthesis, and quantitative real-time PCR (qRT-PCR)

Cells and endometrial tissues were washed three times with PBS and then placed in TRIzol (#15596-018, Invitrogen) to isolate the total RNA following the manufacturer’s protocol. The cDNA was synthesized by reverse transcription-PCR with the PrimeScript RT reagent Kit (#RR047A, Takara). The primer sequences used in these experiments are shown in Additional file 1: Table S4. Amplification was performed in a LightCycler 96 system (Roche, USA) using SYBR GreenTM Master Mix (#RR820A, Takara). The optimal concentration of each primer was determined to ensure high amplification efficiency and specificity in the subsequent qRT-PCR assay. All reactions were performed at least in triplicate. Relative quantification of gene expression was calculated using the 2−∆∆Ct method and normalized to GAPDH as the internal control.

Collagen gel contraction assay

The pretreated wild-type or KAT14-KO immortalized HESCs, as well as the primary human EcESCs, were collected and suspended at a density of 2.0 × 105 cells per 100 µl of medium in 400 µl collagen gel working solution before aliquoting into 24-well plates. The resulting mixture was allowed to gel for 1 h at 37 °C. After polymerization, 1 ml of DMEM/F-12 containing 10% CS-FBS was added on top of the gel lattice and incubated for 24 h. Subsequently, the polymerized gels were released by running a sterile pipette tip along the sides of the well before being cultured for another 24 h in DMEM medium containing 12 ng/ml TGF-β1 or vehicle (PBS) only. The surface area of the contracted gels at 0, 12, and 24 h was measured using Image-Pro Plus v. 6.0 software (Media Cybernetics, Inc., Silver Spring, MD, USA). Measurements of the diameter of each gel were recorded as the average values of the major and minor axes, as previously described [30]. The gel contraction was calculated as follows: diameters of wells – diameters of contracted gels. All reactions were performed at least in triplicate.

Luciferase reporter assay

The wild-type form of the 3′UTR region of the ACTA2 promoter containing the CArG box was subcloned into the pGL3 reporter vector (#E1751, Promega, RRID: Addgene_48743). For the luciferase assay, the pretreated primary human EcESCs were plated on 24-well plates and co-transfected with 0.5 µg of pGL3-α-SMA-pro-Luc or empty (pGL3-Basic) plasmids together with 0.05 mg of pRL Renilla luciferase reporter vector (#E2241, Promega, RRID: Addgene_44379) using Lipofectamine 3000 according to the manufacturer’s instructions. The cells were incubated with TGF-β1 or vehicle (PBS) for 24 h and harvested to investigate the effects of KAT14 on primary EcESC α-SMA expression. The luciferase activity was measured using a dual-luciferase reporter assay kit (#11402ES80, YEASEN) following the manufacturer’s protocol.

In vitro cell migration assay

The migration capability of ESCs was assessed by wound healing assays. In the in vitro wound healing assay, cells were seeded at 2 × 105 cells per well in a 6-well plate and stimulated with 12 ng/ml TGF-β1 or vehicle (PBS) for another 24 h at 37 °C. Subsequently, a scratch was created in the monolayered cell with a sterile pipette tip and then washed three times with PBS. The cells were cultured with FBS-free DMEM/F-12 medium at 37 °C in a humidified 5% CO2 atmosphere. The average width of each scratch was photographed under an inverted microscope and analyzed by Image-Pro Plus v. 6.0 software at 0, 24, and 48 h. Wound healing was measured by calculating the area in µm2 between the lesion edges. The experiment was performed four times.

Antibodies

Detailed information of antibodies used in this study are listed in Additional file 1: Table S5.

Western blot analysis

For total protein extraction, tissues and pretreated cells were washed with ice-cold PBS and incubated with whole-cell lysis buffer NP-40 (#P0013F, Beyotime) supplemented with protein inhibitor phenylmethylsulfonyl fluoride (PMSF, #ST505, Beyotime) and phosphatase inhibitor cocktail (#P5726, Sigma-Aldrich) for 30–50 min on ice and the supernatant was collected by centrifugation (13,000 rpm) at 4 °C for 20 min. Cytosolic and nuclear proteins were separated using a Nuclear and Cytoplasmic Protein Extraction Kit (#P0028, Beyotime). The total histones of primary human EcESCs were extracted using an EpiQuik Total Histone Extraction Kit (#OP-0006-100, Epigentek). Briefly, cells were harvested and washed with ice-cold PBS. The total histones were extracted after successive prelysis, lysis, and addition of balance buffers. The protein concentration of cell lysates was quantified using an Enhanced BCA Protein Assay Kit (#P0010, Beyotime) according to the manufacturer’s instructions. Equal amounts of proteins (20–50 µg/lane for total proteins; 40 µg/lane for cytosolic and nuclear proteins; 40 µg/lane for histone proteins, separately) were separated on 8–18% SDS-PAGE and transferred onto PVDF membranes (#ISEQ00010, Merck Millipore). The PVDF membranes were blocked with 5% skim milk (#D8340, Solarbio) for 2 h at room temperature and then incubated with the corresponding primary antibody, which was diluted in the range of 1:200 to 1:10000 (Additional file 1: Table S5) overnight at 4 °C with shaking. After washing with TBST, the membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature. The protein signals were visualized by chemiluminescence (ECL) substrate (#34,577, Thermo Fisher Scientific) and detected using a Tanon 5200 imaging system (Tanon, Shanghai, China). The protein level was quantified by densitometry with Image-Pro Plus v. 6.0 software and normalized to the level of GAPDH, which served as the internal control to normalize the densitometric intensity. The information on the antibodies applied in this study is shown in Additional file 1: Table S5.

Coimmunoprecipitation (CoIP)

The interaction between endogenous KAT14 and SRF was detected using a Co-IP assay. The primary human EcESCs in primary culture were harvested for immunoprecipitation assays according to standard protocols. Briefly, whole-cell protein was extracted and the cell lysates were centrifuged. The supernatant was collected and precleared by incubation with Protein A/G PLUS-Agarose (#sc-2003, Santa Cruz) and isotypic IgG, followed by incubation with an appropriate amount of specific primary antibody against KAT14 (#sc-398,475, Santa Cruz Biotechnology, RRID: AB_2936359) (4 µg), SRF (#16821-1-AP, Proteintech, RRID: AB_2194384) (2 µg), or normal rabbit IgG (#10284-1-AP, Proteintech, RRID: AB_2877729) (2 µg), separately. Then, resuspended Protein A/G PLUS-Agarose was added to each immunoprecipitation mixture. The beads were washed three times and centrifuged to obtain the indicated protein complexes, which were then resuspended in 1 x electrophoresis sample buffer and separated by SDS–PAGE. Proteins of interest were detected by a subsequent western blot assay with their corresponding antibodies.

Chromatin immunoprecipitation (ChIP)

Enrichment of the target protein at the ACTA2 promoter was analyzed by ChIP assays using a commercially available EZ-ChIP™ Chromatin Immunoprecipitation Kit (#17–371, Merck Millipore) based on the manufacturer’s instruction. Subsequently, the immunoprecipitated DNA fragments were analyzed by quantitative PCR to assess the enrichment of KAT14 at the promoters of ACTA2 and KPNA2, which lacks potential KAT14 binding sites, was employed as a negative control. Additionally, the ChIP assay included PCR to assess the acetylation status of histone H4 at the ACTA2 proximal promoter. The ChIP-qPCR results are presented as enrichment relative to the input. The primers are summarized in Additional file 1: Table S4.

Masson’s trichrome staining

Mouse model samples and human eutopic and ectopic endometrium tissues were fixed in 4% paraformaldehyde (#P1110, Solarbio) at room temperature for 2 h. After washing three times with PBS, the fixed tissues was embedded in paraffin and sectioned into 5-mm slices (5-µm thick). The endometrial morphology was visualized using a Masson’s trichrome staining kit (#G1345, Solarbio) according to the manufacturer’s instructions. The collagen fibers were stained light blue and quantification of fibrotic areas was undertaken by visualizing blue-stained areas using the Olympus IX53 microscope (Tokyo, Japan). Analysis of the endometrium involved two randomly chosen sections per sample, and the areas stained in blue, in proportion to the entire field were calculated using Image-Pro Plus v. 6.0 software.

Immunohistochemistry (IHC) assay

For immunohistochemical staining of cells, approximately 4 × 104 primary ESCs were plated on slides and fixed with 4% PFA. A series of consecutive sections and cell slides were immersed in xylene and ethanol for deparaffinization and hydration, respectively, and then subjected to antigen retrieval by boiling in citrate buffer. Subsequently, the tissue sections and cell slides were incubated overnight with the corresponding primary antibodies at 4°C, followed by immunohistochemical staining (#SAP-9100, ZSGB-BIO). Signal visualization was based on 3,3′-diaminobenzidine (DAB) (#G1212-200T, Servicebio), and cell nuclei were stained with hematoxylin. The images were taken with an Olympus IX53 microscope and analyzed using Image-Pro Plus v.6.0 software to determine the relative expression levels of objective proteins according to the integrated optical density (IOD) of the digital images. At least four randomly selected fields of view were analyzed per sample. The information on antibodies applied in this study is referred to Additional file 1: Table S5.

Immunofluorescence assay

Cell slides and tissue sections were prepared following the aforementioned procedures. For staining, tissue sections and cell slides were permeabilized, and then incubated with the indicated primary antibodies (dilution ratios are provided in Additional file 1: Table S5) at 4 °C overnight. After three washes with 0.1% Tween 20 in PBS, the sections and cells were incubated with the corresponding secondary antibodies, including donkey anti-rabbit (conjugated with Alexa Fluor® 647), donkey anti-goat (conjugated with Alexa Fluor® 568), or goat anti-mouse (conjugated with Alexa Fluor® 488), at room temperature for 1 h. Following cell nuclear staining with an antifade mounting medium with DAPI (#P0131, Beyotime), the samples were mounted and immunofluorescence images were obtained with an Olympus FV3000 confocal microscope (Tokyo, Japan).

Statistical analysis

Statistical analyses were performed using SPSS software version 24.0 (SPSS Inc, Chicago, USA) and GraphPad Prism 8.0.1 software (GraphPad, San Diego, California, USA). All data are shown as the mean ± standard deviation (SD) from at least three independent experiments. Two-tailed Student’s t-tests and the Mann–Whitney U test were employed to compare the quantitative variables between two groups. For experiments involving at least three groups, one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test was used. All sample measurements were performed in a blinded manner. P-values < 0.05 were considered to indicate statistical significance.