Animals, ethics, and BMM culture, identification and polarization
All animal procedures were conducted in accordance with protocols approved by the Experimental Animal Ethics Committee of Guangdong Medical University (no. GDY2003014), and conformed to the Helsinki Declaration of 1975 (as revised in 2008) concerning animal rights. Heterozygous SETD4 KO C57BL/6J mice were bred, mated and genotyped as described previously [20] . Wild type (WT) and SETD4−/− mice (male, 8 weeks old, 18–23 g) were subjected to BMM isolation. BMMs were maintained in Dulbecco’s modified Eagle's medium (DMEM; ThermoFisher Scientific, Shanghai, China) supplemented with L929 cell supernatant (25%, v/v), 20% fetal bovine serum (FBS; ThermoFisher Scientific), 100 U/mL penicillin and 0.1 mg/mL streptomycin, and were incubated in 5% CO2 saturated humidity at 37°C. The culture medium was changed every 2 days. The supernatant of L929 cells was obtained by continuously culturing L929 cells (kindly provided by Cell Bank, Chinese Academy of Sciences, Shanghai, China) in L929 cell special medium (Cat: SCSP-5039, Cell Bank) for 7 days. Undifferentiated BMMs (M0) were subjected to immunofluorescence staining of F4/80 (Cat: 11-4801-82, ThermoFisher Scientific), and their purity was calculated. M0 macrophages treated with 100 ng/mL lipopolysaccharide (LPS; Cat: L2630, Sigma-Aldrich, Shanghai, China) + 20 ng/mL interferon-γ (IFN-γ; Cat: 315-05, PeproTech, Cranbury, NJ, USA) were induced to differentiate into a pro-inflammatory type (M1 polarization), while those treated with 20 ng/mL murine interleukin-4 (IL-4; Cat: 214-4, PeproTech) for 24 h were induced into an anti-inflammatory type (M2 polarization). The M0, M1 and M2 cells were subjected to the following experiments.
Cell counting kit (CCK)-8 and EdU incorporation assays
For CCK-8 assays, M0 cells were seeded onto 96-well plates (8000 cells/well) and allowed to recover overnight. At 0, 1, 2 and 3 day time points, 10 μL of CCK-8 reagents (Cat: SPDA-D010, Beyotime Institute of Biotechnology, Nanjing, China) was added, cells were maintained for additional 3 h, and optical density (OD) at 450 nm was measured using a microplate reader (ThermoFisher Scientific). Each group was duplicated in 4–6 wells. For EdU incorporation assays, cells were added to 96-well plates (10,000 cells/well) and cultured in a 37°C incubator for 24 h before adding EdU DNA proliferation in vitro detection reagents (Cat: C10310, RiboBio, Guangzhou, China), following the instruction of the manufacturer. Fluorescence microscopy was used to image and record the rate of positive cells. The numbers of cells with red fluorescence and Hoechst staining (blue fluorescence) in each field were used to calculate the cell proliferation ratio. Assays of each group were repeated in three wells.
Cell cycle determination by flow cytometry (FCM)
Serum-starved M0 cells were seeded in 6-well plates at a concentration of 5 × 105 cells/well and incubated in L929 supernatant-enriched medium for 12 h, 24 h, 48 h and 72 h. Cells were collected and suspended in 70% ethanol for 24 h at 4°C. After washing with cold phosphate-buffered saline, cells were incubated with RNase A and propidium iodide at 37°C for 30 min. FCM was performed to assess the cell cycle distribution, and data were analyzed using FlowJo 7.6 software (Treestar, Ashland, OR, USA). Each time point was repeated in three wells.
Cell migration assay
M0 cells were serum-starved for 12 h and trypsinized; 4 × 104 cells were collected in a total volume of 200 μL DMEM medium, which was added to the upper Transwell chambers; and 500 μL DMEM medium containing 15% FBS was added to the lower Transwell chambers. A membrane with an 8-μm pore size separated the upper and lower Transwell chambers. After 24 h, the chambers were disassembled. The membranes were fixed in 4% paraformaldehyde for 20 min, stained with 0.1% crystal violet for 15 min, and washed with ddH2O for observation and photography with a microscope. Each cell sample was repeated in three chambers.
Tube-formation assay
Considering that M2 cells are beneficial for tube formation during tissue repair, we tested the contribution of SETD4 to the tube-formation ability of M2 cells. The supernatant was collected from M2 cells derived from WT and SETD4 KO macrophages induced by 20 ng/mL IL-4 for 24 h. Aliquots of 100 μL diluted Matrigel (Corning, NY, USA) were added 48-well plates and incubated at 37°C for 30 min, followed by the addition of 4 × 104 serum-starved human umbilical vein endothelial cells (HUVECs; provided by Dr. XJ Zhang, Guangdong Medical University) and 200 μL of the M2 cell supernatants. After a 4-h incubation, the cells were subjected to microscopic observation and photography, and the formation of tube-like structures by the HUVECs was analyzed by ImageJ software (Version 7, https://imagej. nih.gov/ij/).
RNA isolation, reverse transcription (RT) and PCR
Total cellular RNAs were extracted using TRIZOL reagent (Cat: 15596-026, ThermoFisher Scientific) and quantified by SimpliNano (Biochrom, Germany). Totally 500 ng RNA was used to generate cDNA using an RT kit (Cat: RR047, TaKaRa Beijing, China). Quantitative RT-PCR (qRT-PCR) was performed using a LightCycler 480 II machine (Roche, Basel, Switzerland). The 20-μL reaction mixture comprised 10 µL SYBR Green I PCR Master Mix (#QPK-201, TOYOBO Guangzhou Branch, China), 0.4 µL forward primer (10 µM), 0.4 µL reverse primer (10 µM), 2 µL cDNA, and 7.2 µL ddH2O. PCR amplification was performed as follows: 95°C for 1 min, and 45 cycles of 95°C for 5 s and 60°C for 20 s. The 2−DDCT method was used to determine the levels of mRNAs. Primers spanning the exon–exon junction are listed in Table S1.
Western blotting
Cells were lysed with RIPA buffer (Cat.P0013B, Beyotime Institute of Biotechnology). Samples of total protein (35 µg) were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Merck China, Shanghai). After washing twice with Tris-buffered saline containing 0.1% Tween 20 (TBST), the membranes were incubated with 5% skim milk powder in TBST at 37°C for 1 h and then incubated with primary antibodies at 4°C overnight. The primary antibodies are listed in Table S2. After washing with TBST twice, the membranes were incubated with horseradish peroxidase (HRP)-conjugated IgGs (1:5000; Cat. SA00001-10, Cat. SA00001-2; ProteinTech,Wuhan, China) for 1 h at 37°C. Bands were visualized using enhanced chemiluminescence (ECL) reagents (ThermoFisher Scientific) and analyzed with a gel analysis system (Tanon, Shanghai, China).
Phosphorylation Pathway Profiling Array
The RayBio® Phosphorylation Pathway Profiling Array (Cat.AAH-PPP-1-2, RayBiotech, Guangzhou, China) was used as instructed by the manufacturer to detect the relative levels of phosphorylation of 55 unique proteins from five well-known signaling pathways, including mitogen-associated protein kinase (MAPK), AKT, Janus kinase/signal transducer and activator of transcription (JAK/STAT), nuclear factor (NF)-κB and transforming growth factor (TGF)-β. Briefly, total protein was extracted from M0 cells from WT and SETD4 KO mice. The protein chip membranes were incubated with Blocking Buffer at room temperature for 1 h, and then incubated with 1 mL of a 1:20 dilution of cell lysate overnight at 4°C. The membranes were washed twice with Wash Buffer I & II and then incubated with Detection Antibody Cocktail at 25°C for 2 h. After washing twice with Wash Buffer, the membranes were incubated with HRP-labeled anti-rabbit secondary antibody at 25°C for 2 h. The signals were collected by an ImageQuant LAS4000 Scanner (GE Healthcare Corporation, Pittsburgh, PA, USA) after incubation with an ECL reagent. The original raw data obtained by chip scanning were processed by Raybiotech software for chip background removal and normalization. Differentially expressed proteins (DEPs) were identified by screening for P < 0.05 and a fold-change (FC) in expression of ≤ 0.83, or P < 0.05 and FC ≥ 1.2 [21] .
Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment
Protein function annotation for GO and KEGG pathways were analyzed by the R package “clusterProfiler”, with the standard number of DEPs for a certain GO term/pathway set at ≥ 5 and P < 0.05 (Fisher's exact test). KEGG was used to enrich the DEP-related pathways.
Epidermal growth factor receptor (EGFR) inhibitor treatment of BMMs
The EGFR inhibitor CI-1033 (also known as Canertinib or PD183805, Cat. S1019; Selleck China, Shanghai) was used to treat BMMs. BMMs isolated from WT and SETD4 KO mice were seeded onto 96-well plates and 6-cm dishes and allowed to recover overnight. BMMs were incubated with complete medium supplemented with various concentrations of CI-1033 for 24–48 h. Cells in 96-well plates were subjected to a CCK-8 assay for cell viability, while cells in 6-cm dishes were used for protein extraction and western blotting analysis of phosphorylated (p)-EGFR (Ser1070) and proliferating cell nuclear antigen (PCNA) expression. We used the change in p-EGFR(Ser1070) levels to indicate the activation of ERFR signaling.
Statistical analysis
Statistical analyses were conducted using GraphPad Prism (Version 9.0, GraphPad Software, San Diego, CA, USA). Data are expressed as means ± standard deviation. Differences between two groups were analyzed using umpired Student’s t tests. For comparisons between multiple groups, analysis of variance was used, followed by Tukey’s multiple comparisons test. A P value < 0.05 was considered to be statistically significant.