Antibodies and reagents

Rabbit polyclonal antibodies against H3K27me3, EZH2, NF-κB, JMJD3, Histone H3, and HRP-labelled anti-rabbit IgG were obtained from Cell Signaling Technology, USA, LC3B and β-actin were procured from Abways, China, and Alexa 488 (goat anti-rabbit, Green) was purchased from Servicebio, China. 4,6-Diamidino-2-phenylindole (DAPI), PYO (Cat# P0046), Phorbol 12-myristate 13-acetate (PMA), Triton X-100, glutaraldehyde, and osmium tetroxide were obtained from Sigma-Aldrich, USA. Chloroquine (CQ; Cat# HY-17589A), El1 (Cat# HY-15573), CPI-169 (Cat# HY-15956A), and Curcumin (Cat# HY-N0005) were acquired from MedChemExpress, USA. RPMI 1640 medium was obtained from Gibco, USA; fetal bovine serum (FBS) from PAN-biotech, Germany; and Penicillin–Streptomycin from Solarbio, China. The All-in-One cDNA Synthesis SuperMix was purchased from TransScript, China; the 2xSYBR Green RT-qPCR Master Mix from Thermo Scientific, USA; and the Nuclear and Cytoplasmic Protein Extraction Kit from Beyotime, China. The UNIQ-10 Column Total RNA Purification Kit was obtained from Sangon Biotech, China; the SimpleChIP® Enzymatic Chromatin IP Kit from Cell Signaling Technology, USA; and Protein A/G PLUS-Agarose beads from Santa Cruz Biotechnology, USA. Additional reagents included RIPA lysis buffer, phosphatase inhibitor, PMSF, and BSA (all from Beyotime, China), a protease inhibitor cocktail (Bimake, China), a BCA Protein Assay Kit (KeyGen Biotech, China), DMSO (Sigma-Aldrich, USA), the Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, USA), and EMbed 812 resin (Electron Microscopy Sciences, USA). Unless otherwise specified, all other reagents were of analytical grade.

Cell lines and cell culture

THP-1 cells (Chinese academy of Sciences, Cat# SCSP-567), a human acute monocytic leukemia cell line, were selected for analysis in this study. The cells were cultured in RPMI 1640 medium (Gibco, Cat# 11875101) containing 12% FBS (PAN-biotech, Cat# P30-3302; inactivated at 56 °C for 30 min) and antibiotics (100 U/mL Penicillin and 100 µg/mL Streptomycin; Solarbio, Cat# P1400), and incubated at 37 °C in a 5% CO2 and humidity-saturated environment. Suspension THP-1 monocytes were seeded in well plates and differentiated into adherent THP-1 macrophages by treating with 100 ng/mL PMA (Sigma-Aldrich, Cat# 79346) for 48 h, followed by a 24-h incubation in RPMI-1640 medium without PMA.

For PYO treatment, THP-1 macrophages were treated with 50 μM PYO for 6 h. In subsequent experiment, cells were treated with 10 μM CQ for 12 h, 1 nM CPI-169 for 24 h, 1 μM EI1 for 24 h, or 10 μM Curcumin for 12 h, depending on the experimental design. DMSO was used as a vehicle control at a final concentration of 0.1% for all treatments.

RNA extraction and RT–qPCR analysis

Total RNA from THP-1 macrophages was extracted using the UNIQ-10 Column Total RNA Purification Kit (Sangon Biotech, Cat# B511361), following the manufacturer’s instructions. RNA purity and concentration were assessed with a Nano Spectrophotometer (Implen, Germany). cDNA was synthesized using All-in-One cDNA Synthesis SuperMix (TransScript, Cat# ABIN5519497), and amplification was carried out in a RT–qPCR instrument (Bio-Rad CFX96, USA) following the Maxima SYBR Green qPCR Master Mix protocol (Thermo Scientific, Cat# K0252). ACTB was set as an internal reference, and the relative quantification of target gene expression was determined by the 2−ΔΔCt method, with normalization to ACTB. The relative gene expression levels in each experimental group were normalized to that of the control group. The specific primer sequences used for RT–qPCR are listed in Supplementary Table 1.

Protein extraction and co-immunoprecipitation (Co-IP)

THP-1 macrophages were lysed using RIPA buffer (Beyotime, Cat# P0013B) containing protease inhibitors (Bimake, Cat# B14012), phosphatase inhibitors (Beyotime, Cat# P1081), and PMSF (Beyotime, Cat# ST506). The lysates were centrifuged at 12000 × rpm for 15 min at 4 °C, and the supernatant was collected for protein extraction. Primary antibodies against H3K27me3 (1:50; Cell Signaling Technology, Cat# 9733, RRID: AB_2626029) and NF-κB (1:100; Cell Signaling Technology, Cat# 8242; RRID: AB_10859369) were added to the protein solution and incubated at 4 °C overnight, followed by incubation with Protein A/G PLUS-Agarose beads (Santa Cruze, Cat# sc-2003) for 2–4 h. The protein–antibody–bead complexes were gently washed three times with RIPA buffer, then denatured by boiling for the subsequent Western blot analysis.

Western blot

For Western blot analysis, THP-1 macrophages were lysed using RIPA buffer for protein extraction. Protein concentrations were determined using a BCA Protein Assay Kit (KeyGen Biotech, Cat# KGP903). Samples were denatured by boiling for 5 min, and equal amounts of protein (20 µg/well) were loaded onto SDS–PAGE gels. After electrophoresis, proteins were transferred onto PVDF membranes and blocked with 5% BSA (Beyotime, Cat# ST023) for 2 h at room temperature. Membranes were then incubated overnight at 4 °C with primary antibodies against H3K27me3 (1:1000), EZH2 (1:1000; Cell Signaling Technology, Cat# 5246; RRID: AB_10694683), NF-κB (1:1000), JMJD3 (1:1000; Cell Signaling Technology, Cat# 3457; RRID: AB_1549620), H3 (1:2000; Cell Signaling Technology, Cat# 4499; RRID: AB_10544537), LC3B (1:2000; Abways, Cat# CY5992), and β-actin (1:10000; Abways, Cat# AB0035). After washing with TBST, membranes were incubated with HRP-labelled Anti-rabbit IgG (1:3000; Cell Signaling Technology, Cat# 7074; RRID: AB_2099233) at room temperature for 2 h. The immunoreactive bands were visualized by enhanced chemiluminescence (Merck Millipore, Cat# WBKLS0500) and imaged using a ChemiDoc™ MP Imaging System (Bio-Rad, USA).

Immunofluorescence

THP-1 macrophages were seeded onto coverslips. After washing with PBS, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature and then permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, Cat# T8787) for 15 min. Following three washes with PBS, cells were blocked with 5% BSA for 1–2 h. The coverslips were incubated overnight at 4 °C with primary antibody against LC3B (1:200), and then with Alexa Fluor® 488-conjugated Goat anti-rabbit IgG (1:500; Servicebio, Cat# GB25303; RRID: AB_2910224) for 1 h at room temperature in the dark. Nuclei were stained with DAPI (Sigma-Aldrich, Cat# D9542) for 5 min. Finally, the samples were examined under a fluorescence microscope (Olympus FV-10000, Japan).

Transmission electron microscopy

THP-1 macrophages were fixed in 0.1M PBS with 2.5% glutaraldehyde (Sigma-Aldrich, Cat# G5882) and 2% paraformaldehyde at room temperature for 2 h, followed by post-fixation with 1% osmium tetroxide (Sigma-Aldrich, Cat# 201030) for 2 h. Then, dehydrated in a graded series of ethanol, the cells were embedded in EMbed 812 (Electron Microscopy Sciences, Cat# 149000) and sectioned into ultra-thin slices of about 70 nm. The sections were subsequently double-stained with 3% uranyl acetate and lead citrate, and the autophagy structures were observed under transmission electron microscopy (EM900, Carl Zeiss, Oberkochen, Germany).

Nuclear and cytoplasmic protein extraction

PMSF was added to both the cytoplasmic (Beyotime, Cat# P0028) and nuclear protein extraction reagents (Beyotime, Cat# P0028) to achieve a final concentration of 1mM. Cells were harvested using a scraper and centrifuged at 500 g for 3 min. To each 20 μL cell pellet, 200 μL of cytoplasmic protein extraction reagent was added. The mixture was vortexed for 15 s, chilled for 10 min, vortexed again at maximum speed for 10 s, and then centrifuged at 14,000 g at 4 °C for 10 min to separate cytoplasmic proteins from the supernatant, which was immediately transferred to pre-cooled tubes. The remaining pellet, containing the nuclei, was resuspended in 50–100 μL of nuclear protein extraction reagent, vortexed and chilled as before, and centrifuged to collect nuclear proteins in the final supernatant for further analysis or storage.

Chromatin immunoprecipitation (ChIP)

ChIP–qPCR was performed with SimpleChIP® Enzymatic Chromatin IP Kit (Cell Signaling Technology, Cat# 9003) according to the manufacturer’s protocol. THP-1 macrophages were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by chromatin digestion using Micrococcal nuclease (0.5 μL per tube) and fragmentation into 150–900 bp segments via sonication (Pico, Belgium) for 12 cycles (5 s on/off). Target primary antibodies against H3K27me3 (1:50) and NF-κB (1:100) and Normal IgG were added to the corresponding ChIP samples. Purified DNA was amplified using Maxima SYBR Green qPCR Master Mix. Data were represented as the percentage of input DNA. The primer sequences used for amplifying the gene promoter regions in ChIP–qPCR are detailed in Supplementary Table 2.

Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 8 software (GraphPad, USA). Group comparisons were conducted using the t test or one-way analysis of variance (ANOVA) with Bonferroni post hoc tests. Differences were considered statistically significant when the P value was less than 0.05.