Reagents and plasmids
Antibodies against HDAC3 (#85057, 1:1000 for western blot; #3949, 1:100 for immunofluorescence staining), HDAC1 (#5356, 1:1000), HDAC2 (#5113, 1:1000), HDAC8 (#66042, 1:1000), CTSB (#31718, 1:1000 for western blot; 1:100 for immunofluorescence staining), CTSD (#69854, 1:1000), RIP1 (#3493, 1:1000), GAPDH (#2118, 1:2000), β-ACTIN (#4970, 1:2000), Phosphorylated-RIP1 (#38662, 1:1000), Phosphorylated-P65 (#3033, 1:1000), P65 (#8242, 1:1000 for western blot; 1:100 for immunofluorescence staining), H3K27ac (#8173, 1:50 for CHIPseq) were purchased from Cell Signaling Technology (CST). Leupeptin (#S7380), CA-074 (#S7420) were bought from Selleck. MG132 (#M8699) was bought from Sigma-Aldrich. TNFα (#410-MT-010) was bought from R&D systems. Lipo3000 (InvitroGen, #L3000015) was used for the transfection experiment. PCMV-C-EGFP (#D2626) was bought from Beyotime Biotechnology. The plasmids needed for the experiment were synthesized by GENEWIZ.
Cell and lenti-virus
RAW264.7 (murine monocytic cell line, ATCC: TIB-71), 293T and Neuro-2a cells were cultured in Dulbecco’s-modified Eagle’s medium (DMEM) (Gibco) containing 10% fetal bovine serum (FBS) (Peak) and 50 units/ml penicillin/streptomycin (Gibco). BMDMs were generated from the bone marrow of 8-week-old mice and cultured in RPMI-1640 medium (Gibco) with 10% FBS and recombinant mouse granulocyte–macrophage colony-stimulating factor (PeproTech, 50 ng/ml).
CRISPR/Cas9 system was used for construct of Hdac1−/−, Hdac2−/−, Hdac3−/− and Hdac8−/− RAW264.7 cells. The sequences of small guide RNA (sgRNA) were listed in Additional file 1: Table S1. sgRNA was synthesized and ligased into lenti-V2 vector. And both sgRNA loaded lenti-V2 vector, pMD2.G (Addgene, #12259) and psPAX2 (Addgene, #12260) were then transfected into 293T cells by Lipo3000 to obstain Lentivirus. Lentivirus containing supernatant was collected and used for infecting RAW264.7 cells. After 48 h infection, RAW264.7 cells were then selected with 4 μg/ml puromycin (InvivoGen, #ant-pr-1) in DMEM complete medium for one week. Flow cytometry was used to separate the cells into monoclonal and western blot technology was used to identify whether the gene was knocked out.
Quantitative real-time PCR (RT-qPCR)
RNA of 2 × 106 cells seeded on six-well plates was extracted by NucleoZol (MNG). Briefly, 600 μl NucleoZol was added with 240 μl ddH2O and then centrifugated at 12,000g, 4 ℃ for 15 min, and 700 μl supernatant was added with 700 μl isopropanol. After centrifugation, the RNA precipitation was washed by 75% ethanol for two times. PrimeScript™II 1st Strand cDNA Synthesis Kit (Takara, #6210A) was used for Reverse transcription of RNA into complementary DNA (cDNA). And SYBR Green (Takara) was used for qPCR. All the steps are done according to the manufacturer’s instructions. The sequences for primer pairs are listed in Additional file 1: Table S2. RNAseq was done by the company (Novogene) for database construction and sequencing.
Western blot
Cells were lysed for 30 min on ice by lysis buffer (Beyotime Biotechnology, #P0013) which contained phosphatase inhibitor cocktail (Roche, #04906845001) and complete protease inhibitor cocktail (Roche, #04693132001). After centrifugation at 12,000g, 4 ℃ for 15 min and boiled with 4X Protein SDS PAGE Loading (Takara, #9173) for 10 min, the proteins were separated through SDS-PAGE gel and transferred onto PVDF membrane. The membrane was blocked with 5% skimmed milk for 2 h, and then incubated with interested antibodies at 4 ℃ overnight. After stained with HRP-coupled secondary antibodies for 2 h, the proteins on membrane were then detected by Chemiluminescence system (Bio-Rad ChemiDoc MP).
Chromatin immunoprecipitation (ChIP)
Chromatin IP Kit (CST, #9005) was used for Chromatin Immunoprecipitation. All process was done according to manufacturer’s protocol. In brief, RAW264.7 cells in 20 ml DMEM complete medium were added with 540 μl 37% formaldehyde for 10 min at room temperature and then added with 2 ml 10X glycine for 5 min. After washed by 20 ml cold PBS for 3 times, cells were subject to micrococcal nuclease digestion and sonication through which DNA was processed to the length of approximately 150–900 bp. Chromatin concentration was measured using 10% of the sample after DNA purification. 10 μg chromatin was then mixed with indicated antibodies at 4 ℃ overnight. 30 μl Protein G Magnetic Beads were added to each IP reaction and incubate for 2 h at 4 ℃. After washing in low salt buffer for 3 times and in high salt buffer for 1 time, beads were resuspended with ChIP Elution Buffer. After eluted from beads through vortexing (1200 rpm) at 65 ℃ for 30 min, Chromatin was reverse crosslinked with RNase and proteinase K at 65 ℃ for 2 h. Then DNA solution was then subjected for library establishment after DNA purification.
KAPA HyperPlus Kit (KAPA Biosystems, #KK8514) was used for Library establishment. All process was done according to manufacturer’s protocol. In brief, 50 μl sample was subjected to End Repair and A-tailing at 65 ℃ for 30 min. And then the sample was ligased with different adapter at 20 ℃ for 15 min. The sample was mixed with 88 μl KAPA Pure Beads at room temperature for 15 min and then placed on a magnetic frame. After washed by 80% ethanol for 3 times, DNA was amplified with following PCR program:98 ℃, 45 s; then 8 cycles of 98 ℃, 15 s, 60 ℃, 30 s and 72 ℃, 30 s; 72 ℃, 1 min; 4 ℃, ∞. The sample was again mixed with 88 μl KAPA Pure Beads at room temperature for 15 min and then placed on a magnetic frame. After washed by 80% ethanol for 3 times, DNA was ready for sequencing by company (Novogene).
Immunofluorescence staining
RAW264.7 cells seeded on Glass Bottom Dish (Cellvis, #D29-10-1.5-N) were fixed using 4% paraformaldehyde for 15 min, permeabilized by 0.1% Triton X-100 (Sigma-Aldrich, #T9284) for 10 min, and blocked with 1% BSA (Beyotime Biotechnology, #ST023) for 1 h at room temperature. They were then stained with anti-CTSB antibodies (CST, #31718, 1:100), anti-HDAC3 antibodies (CST, #3949, 1:100) or anti-P65 antibodies (CST, #8242, 1:100) overnight. After washed by PBS three times, they were followed reacted with Alexa Fluor 488-conjugated anti-mouse IgG secondary antibody (life technologies, #A11001, 1:1000). The nuclei were stained with DAPI (Beyotime Biotechnology, #C1002, 1:1000).
Neuro-2a cells seeded on Glass Bottom Dish were transfected with 1.5 μg PCMV-C-EGFP-RIP1 for 24 h and CA-074 was added at 12 h after transfection. Then the cell culture medium was removed and lyso-tracker (Solarbio, #L8010, 1:15,000) staining solution was added for 1 h at 37 ℃. Cells were washed by PBS for 3 times and The nuclei were stained with Hoechst (Beyotime Biotechnology, #C1027, 1:100).
Cells were imaged on a confocal microscope (Nikon Tl-S).
Mouse acute lung injury (ALI) model
Hdac3f/f mice on a C57BL/6 J background were bought from the Jackson Lab (Stock number: 024119). The Lysm-Cre mice were bought from the Jackson Laboratory (Stock number: 004781). All mice were bred in Animal center of Suzhou Institute of Systematic Medicine. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Suzhou Institute of Systems Medicine (ISM-IACUC-0019-R). Age-matched male LysmCre and LysmCreHdac3f/f littermates were used for all experiments. In LPS-mediated acute lung injury model, 5 mg/kg LPS was injected into the airway of mice. After 12 h, the mice were executed for BALF extraction. In pseudomonas aeruginosa-mediated acute lung injury model, the mice were intratracheal instillation of a dose of pseudomonas aeruginosa (2 × 107 CFU/per mouse). The survival rate was observed every 12 h up to 120 h. The temperature of mice was monitered until death appears. BALF extraction and lung tissue collection were done in 36 h after pseudomonas aeruginosa infection.
Bronchoalveolar lavage fluid (BALF) extraction and analysis
An incision in the bronchus of mice was cut with scissors after anesthesia. Then the syringe was used for injecting 1 ml cold PBS into the airway and about 70–80% PBS was withdrawn. After twice the same steps, about 1.5 ml BALF was obstained. Centrifugation was performed to separate the cells and supernatants for inflammatory factors assay.
Enzyme-linked immunosorbent assay (ELISA)
IL-6 (eBioscience, #85-88-7066-77), TNFα (eBioscience, #85-88-7324-88) and HMGB1 (IBL, #ST51011) were detected with ELISA kits according to the manufacturer’s protocols.
Histological analysis
The scissors were used to open the thoracic cavity of mice and the syringe was used to collect the blood from heart to avoid blood contamination after anesthesia. Then the lung was resected and fixed in 4% paraformaldehyde (PFA). The samples were then embedded with paraffin and sliced into 4 um sections for haematoxylin and eosin (H&E) staining. The injury score was calculated according to previous research [34].
Statistical analysis
Data are presented as mean ± SEM. The significance of difference between groups was detected by two-tailed Student’s t-test or one-way analysis of variance test as appropriate. Mouse survival curve analysis was calculated by the log rank test. GraphPad Prism5 was used for data analysis. P-value < 0.05 was considered statistically significant.