RAW 264.7 cells (American Type Culture Collection) were cultured in DMEM with 10% fetal bovine serum (Gibco by Life Technologies). QKI silencing and overexpression in RAW264.7 cells were achieved as described before . Here, cells were represented as SC (Scramble), shQKI and QKI. Mouse peritoneal macrophages were obtained as described previously . Three days before collecting peritoneal cells, 6- to 8-week-old mice were injected with 1 ml of 3% thioglycolate medium. Cells were further enriched by discarding the culture medium containing non adhesive cells after seeding into 6-well plates for 2 h. Bone marrow-derived macrophages (BMDM) were harvested as previously described. In brief, bone marrow cells were collected from femur of male QKIflox/flox and QKIflox/flox-CreLySM mice. Then these cells were seeded into plastic petri dish in complete RPMI 1640 medium supplemented with 20 ng/ml recombinant murine macrophage colony stimulating factor (M-CSF, Peprotech, Rock Hill, USA) for 7 days. Following that, adherent cells were washed with PBS, and cellular infection was carried out through incubating with live S. aureus for indicated time.
Cloning of RNF6 and DNA plasmids constructs
RT-PCR was used to isolate full-length cDNAs of Rnf6 encoding mouse on mRNA prepared from whole Raw264.7 cells, then cDNAs was subcloned into pCS2-MT vector with endonuclease sites of NotI and ApaI.
RAW 264.7 cells were seeded in 6-well plates at a density of 7 × 105 cells/well in antibiotic-free DMEM, and were transfected with 50 nM of siRNA (Genepharma, China) using the transfection reagent lipofectamine 2000 (Thermo Fisher, USA). After 48 h of transfection, the cells were infected with bacteria for indicated time. The cells were then used for the Western blot and the supernatants were collected for phagocytosis analysis. The siRNA sequences were as follows:
siQKI 5′-GAACAAAGAAACCCUUUAUTT-3′ (forward) and 5′-AUAAAGGGUUUCUUUGUUCTT-3′ (reverse); siLC-3 5′-GCUCCUGAUCUGCUAAUAATT-3′ (forward) and UUAUUAGCAGAUCAGGAGCTT-3′ (reverse); siRnf6-1 5′- GCUAAUGAGAGACCAUAAUTT-3′ (forward) and AUUAUGGUCUCUCAUUAGCTT-3′ (reverse); siRnf6-2 5′- GCAAAUAGAACCCGAUCUATT-3′ (forward) and UAGAUCGGGUUCUAUUUGCTT-3′ (reverse); siOtulin 5′- GGAUAUCAGAACCCAGGUUTT-3′ (forward) and AACCUGGGUUCUGAUAUCCTT-3′ (reverse); siMsl-1 5′- GCACUUCAUGGGUUAUCAUTT-3′ (forward) and AUGAUAACCCAUGAAGUGCTT-3′ (reverse); Negative control 5′-UUCUCCGAACGUGUCACGUTT-3′ (forward), 5′-ACGUGACACGUUCGGAGAATT-3′ (reverse);
Male C57BL/6 mice [8–10 weeks old] were purchased from the Experimental Animal Center of Fourth Military Medical University. QKI conditional knock (LysM+QKIfl/fl) mice used were generated in the same way as Wang described . In brief, heterozygous QKI-floxed transgenic (QKI fl/wt) mice were gener-ated. These mice were backcrossed with C57BL/6 mice (Animal Center of the Fourth Military Medical University) for 11 generations under specific, pathogen-free conditions. Heterozygous breeding pairs were used to generate homozygote QKI fl/fl mice. QKI conditional knock mice (LysMCre QKIfl/fl) were generated byserial breeding of QKI fl/fl mice with mice that have a Cre recombinase controlled by a LysM promoter, in which the conditional QKI allele is excised in myeloid cells. Age-matched QKIfl/fl and LysMCre QKIfl/fl (mice were heterozygous for LysM Cre) male mice were used in all experiments. All mouse experiments and procedures were approved by the Laboratory Animal Center of Fourth Military Medical University and conducted in conformity with the ethical standards. Mice were housed at the animal care facility at 22 °C with 12-h light/dark cycles. All mouse experiments and procedures were approved by the Laboratory Animal Center of Fourth Military Medical University.
Bacterial strains and phage used
Methicillin-resistant Staphylococcus aureus (MRSA) strain Mu50 / ATCC 700699 was gift from Northwest A&F university. They were grown in LB media. The cultures were grown overnight, followed by subculture until logarithmic phase (A600 nm = 0.8).
Induction sepsis model
C57BL/6 J male or transgenic mice [6–8 weeks] were used. Mice were injected intraperitoneally with live S. aureus / ATCC 700699 (1 × 109 or 1.5 × 109 CFU per mouse) or pyrogen-free phosphate-buffered saline (PBS) alone. Body temperature was measured using a rectal thermometer at various time points after infection. Lung, spleen, liver, and kidney were collected and homogenized at 24 h after infection. The homogenates were serial diluted in PBS and plated on tryptic soya agar plates. Plates were incubated at 37 °C overnight and the number of colonies were calculated. For survival studies, mice were observed for 3 days following infection. Death of mice was recorded and the data were analyzed for statistical significance of differences between groups.
Total RNA was extracted with Trizol reagent (Life Technologies) and2μg of total RNA was reverse transcribed with M-MuLV reverse transcriptase (Takara) and mixture of random and oligo-dT primers.
Real-time PCR were performed with universal SYBR Green PCR Master mix on BioRad CFX96 Real-Time PCR Detection System (BioRad) with specific primers.
TLR2 5 ′-GCAAACGCTGTTCTGCTCAG-3′ (forward) and 5 ′-AGGCGTCTCCCTCTATTGTATT-3′ (reverse);
Dectin1 5 ′-GACTTCAGCACTCAAGACATCC-3′ (forward) and 5 ′-TTGTGTCGCCAAAATGCTAGG-3′ (reverse);
QKI (mouse) 5´-TAGCAGAGTACGGAAAGACATG-3´(forward) and 5´-GGGTATTCTTTTACAGGCACAT-3´(reverse);
β-actin (mouse) 5´-GTGACGTTGACATCCGTAAAGA-3´(forward) and
QKI (human) 5´-AGAGCAGTTGAAGAAGTGAAG-3´(forward) and 5´-AGAAGGTCATAGGTTAGTTGCC-3´(reverse).
β-actin (human) 5′- GGCTACAGCTTCACCACCAC -3′ (forward) and 5′- TGCGCTCAGGAGGAGC -3′ (reverse).
Protein lysates were harvested from macrophages using lysis buffer containing 50 mM Tris–Cl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA-free protease inhibitor cocktail (Roche) and 1 mM phenylmethylsulfonyl fluoride (PMSF). 50 μg of lysates were separated by SDS-PAGE and transferred to PVDF membrane (Millipore) for western blot analysis with specific primary antibodies (1:1000) for Ubiquitin (abcam, cat#ab13493), Msl2(CST,cat#ab44006), Outlin (abcam, cat# ab211328), p62 (Cell Signaling Technology, cat#ab16177), PI3Kp110α (Cell Signaling Technology,cat#ab4255), PI3K-p110β(Cell Signaling Technology,cat#ab3011), PI3Kp110δ(Cell Signaling Technology,cat#ab5405), Vps34(Cell Signaling Technology,cat#ab4263, AKT(Cell Signaling Technology,cat#ab4685), p-AKT(Ser473) (Cell Signaling Technology,cat#ab4060), mTOR(Cell Signaling Technology,cat#ab2972), Phospho-mTOR (Ser2448) (Cell Signaling Technology,cat#ab5563) and LC3 (Cell Signaling Technology, cat#ab4108), QKI (Sigma), LC3-II(Cell Signaling Technology, cat#ab2775), flag(Sangon, cat#D191041), HA(Sangon, cat#D191044), and Myc(Sangon, cat#D199941), Rnf6 (Thermo, cat#PA5-59044), EDC4 (proteintech, cat#D17737-1-AP) and β-actin(Sangon, cat#D191047). Immunolabelled proteins were detected by using appropriate HRP-conjugated secondary antibodies (Thermo, cat#31460), followed by visualization with ECL (Sangon).
Cells were lysed in IP buffer (50 mM Tris–Cl, pH 7.5, 150 mM KCl, 0.5% NP40, 1 mM PMSF) at room temperature for 15 min with rotating. The lysate was centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was collected and incubated with appropriate antibodies overnight at 4 °C. Then the mix of supernatant and antibodies was immobilized on Pierce A/G magnetic beads (Thermo) at 4 °C overnight with rotating. The beads were subsequently washed three times with IP buffer. For detection of polyUb chains on QKI, beads were washed with IP buffer supplemented with 0.1% SDS. The bound material was fractionated by SDS-PAGE followed by Western blot analysis.
Protein extraction and iTRAQ mass spectrometry analysis
RAW264.7 cells were seeded in 10-cm dishes and transfected with Flag-QKI or Control vector for 36 h, followed by bacteria infection for 4 h in vitro. The analysis procedures in the same way as Zhao described . The cells were harvested and lysed in complete lysis kit (Roche) with protease and phosphatase. Each sample (100 mg of protein) was digested with trypsin solution and labeled with the iTRAQ reagents (Applied Biosystems) with 113, 114, 115, or 116 reporter ions according tothe manufacturer’s protocol. Subsequently, the labeled peptides were mixed equally and separated by 1260 Infinity HPLC (Agilent Technologies), followed by nano liquid chromatography tandem mass spectrometry using the Hybrid Quadrupole-Orbitrap mass spectrometer (Q-Exactive; Thermo Fisher Scientific) equipped with a nano-UPLC RSLC Ultimate 3000 (Dionex). Both peptide identification and quantitation were performed in an overall workflow in Proteome Discoverer software (version 1.4; Thermo Fisher Scientific) and searched against the UniProt human canonical sequence protein database (October 7, 2011; 56,869 entries) using Mascot search engine (version 2.4). For protein identification, 95% confidence was used. For quantitation and further validation experiments, all reported data were based on 95% confidence for protein identify cation as determined by Proteome Discoverer (Unique peptide > 1).
Dual-luciferase reporter assay
3′UTR of PI3K-p110β was introduced in to psiCHECKTM-2 reporter (Promega) with endonuclease sites NotI and XhoI, and complementary sequence mutation sites of seed sequence was designed on PI3K-p110β-3′UTR(P-3′UTR) to establish PI3K-p110β-mutant (P-3′UTR MUT). The dual luciferase reporter plasmids P-3′UTR and P-3′UTR were co-transfected with QKI or control vector respectively into RAW264.7 cells. Cells were harvested and lysed 48 h after transfection, and the dual-luciferase reporter assay kit (Beyotime Biotechnology Co., Ltd., Shanghai, China) was used to detected Renilla luciferase assay and firefly luciferase separately. The Renilla luciferase served as internal control. The transfection experiments were performed in triplicate for each plasmid construct.
Raw264.7 cells or peritoneal cells were seeded into a 24-well plate (2 × 105 cells/well) (Corning, USA) followed by co-cultured with MRSA at the ratio of 1:50 for 1 h. Then gentamicin (30 μg/mL, MedChem Express, USA) was added into wells to kill extracellular bacteria. For phagocytosis assay, cells were lysed and plated onto LB media. The number of bacteria colonies was counted after 12 h. For assessing the bactericidal ability of macrophages, after incubated with gentamicin, cells were cultured for another 8 h to allow intracellular bacteria killing. Then cells were lysed and plated onto LB media to count bacteria colonies 12 h later.
For detection of phagocytosis of RAW264.7 cells using Phagocytosis Assay Kit (IgG FITC) (NO.500290, USA), RAW264.7 cells (2 × 105cells/ml) plated on a 4 well chamber slide and allowed to adhere overnight. Latex beads-rabbit IgG-FITC complex was added directly to culture medium at a 1:200 dilution and incubated at 37 °C for 2 h. Cells were gently washed with assay buffer twice, followed by counterstaining with 40 μM Hoechst 33342 for 10 min at 37 °C. After two washes, cells were visualized at 20× magnification with a microscope (Olympus IX71). The inhibitor of ULK1(SBI-0206965) and NOX2(GSK2795039) use in this study were purchased from MCEMED EXPRESS.
Nuclear and cytoplasmic protein extraction
RAW 264.7 cells (1 × 107) were incubated with bacteria for indicated time, remove bacteria and wash by PBS for five times. The cells were harvested and nuclear and cytoplasmic protein was extracted by Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, China).
RAW 264.7 cells (1 × 107) were stimulated with bacteria for 6 h and harvested and re-suspended in 1.28 M sucrose, 40 mM Tris–HCl at pH 7.5, 20 mM MgCl2, and 4% Triton X-100 for 20 min on ice. Cells were centrifuged at 2500×g for 15 min and the cell pellet was lysed in RIP buffer (150 mM KCl, 25 Mm Tris at pH 7.4, 5 mM EDTA, 0.5 mM DTT, 0.5% NP40) and centrifuged at 13,000 rpm for 10 min. Then the rabbit anti-QKI antibody (Bethyl Laboratories) or rabbit IgG (Bethyl Laboratories) was added to the supernatants and incubated for 2 h at 4 °C. Then protein A/G PLUS-Agarose was added and incubated for 1 h at 4 °C. After washing with RIP buffer three times, the complexes were incubated with 0.5 mg/ml proteinase K at 55 °C for 15 min. Trizol (Life Technologies) was added to extract the RNA. Reverse transcription was carried out and real-time PCR was performed using the following specific primers for PI3K-p110β: 5 ′-GATTATGTTGAACTTATTATTC-3′ (forward) and 5 ′-ATATTATATTTGCCCCACCAAT-3′ (reverse). The level of β-actin mRNA in each immunoprecipitation sample was used to normalize to the RIP results.
RNA fluorescence in situ hybridization
FAM labelled PI3K-p110β probe (5′-TAGAAGATGAACTGCCCCGC-3′) and negative control (5′-TTTCAGATGTAGGCAAGCC-3′) were purchased from Sangon (Shanghai, China). Firstly, cells were cultured and fixed in 4% paraformaldehyde for 20 min at room temperature, followed with PBS washing three times. Cells were permeabilized with proteinase K (20 μg/ml) for 5 min at 4 °C (Beyotime, Shanghai, China), followed with PBS washing three times. Then cells were incubated with SSC buffer (Solarbio, Beijing, China) for 1 h at 37 °C. Next, FAM labeled PI3K-p110β probe mix buffer was added in the dark followed with incubated at 55 °C overnight. Remove the probe mix buffer, cells were washed with 2 × SSC buffer for 10 min, 1 × SSC buffer for 10 min, 0.5 × SSC buffer at 37 °C. Cells were gently washed with assay buffer twice, followed by counterstaining with 40 μM DAPI (Sangon, Shanghai, China) for 10 min at 37 °C. After two washes, cells were visualized at 20× magnification with a microscope (Olympus IX71).
Indirect immunofluorescence analysis
RAW 264.7 cells were grown on coverslips overnight. MRSA were incubated with FITC-conA (Sangon, Shanghai, China) for 30 min, followed with PBS washing three times. Then cells were incubated with MRSA for 1 h. PBS-washed cells were fixed with 4% formaldehyde for 20 min and then permeabilized with 0.1% Triton X-100 (Sangon, Shanghai, China) for 15 min. After blocking with 5% goat serum for 1 h, cells were incubated with 1:1000 dilution of various antibodies overnight. After washing, cells were incubated with a 1:1000 dilution of Day Light 594 conjugated goat anti-rabbit IgG (TermoScientifc) for 30 min and counterstained with DAPI (Sigma, USA) to stain cell nuclei. Microscopy (Olympus IX71) was used for observing cells and Cell Sens imaging software was used to capture the images.
Transmission electron microscope
Cultured macrophages were fixed in sodium cacodyl ate-buffered (0.1 mol/L, pH 7.4) 2.5% glutaraldehyde solution for 2 h, then rinsed (3 × 10 min) in sodium cacodylate-buffered (0.1 mol/L pH 7.4) 7.5% saccharose and post-fixed in 1% OsO4 solution for 1 h. After dehydration in an ethanol gradient (70% ethanol for 20 min, 96% ethanol for 20 min, 100% ethanol for 2 × 20 min), samples were embedded in Durcupan ACM and then were stained with uranyl acetate and lead citrate. Sections were examined under a FEI TECNAI spirit (USA) microscope at 100 kV.
Cytokine and ROS detection
The level of TNF-α, IL-1β, IL-10 and IL-6 were measured using ELISA kits (Dakewe, Shenzhen, China) according to the manufacturer’s instructions. The intracellular level of reactive oxygen species was determined using 2′, 7′-dichlorofluorescein diacetates (DCFH-DA, Sigma, USA) according the manufacturer’s instruction. In brief, cells (106 cells/well in 6-well plates) were cultured with S. aureus for 1 h and incubated with DCFH-DA at 37 °C for 30 min. The green fluorescence of 2′7 − dichlorofluoresce (DCF) was recorded at 515 nm using a FACS Vantage system (Becton–Dickinson, San Jose, CA, USA), and 10 000 events were counted per sample.
Nanoparticle for QKI siRNA delivery
QKI siRNA was synthesized by Sangon Biotech Company (Shanghai, China). And specific gene silencing nanoparticles named as siQKI-LCP (lipid-coated particles) was achieved as described previously .
Human PBMC and monocytes isolation and analysis
The samples of healthy controls and Patients with sepsis were collected from Xi Jing hospital of the Fourth Military Medical University (Xi’an, China). Patients with sepsis meeting SIRS criteria (Any 2 of the Following: Heart rate > 90 beats/min, Respiratory rate, beats/min > 20, Temperature > 38 or < 36 °C, White blood cell count > 12,000/mm3 or 10% bandemia).Written informed consent from a next-of-kin was required for enrolment. Retrospective consent was obtained from patients, if possible.
According to the clinical observation and diagnosis, inclusion criteria were proven bacterial infection, together with a systemic inflammatory response (two signs or more among increased heart rate, abnormal body temperature, increased respiratory rate and abnormal white-cell count) and acute organ dysfunction and/or shock.
EDTA-anticoagulated blood samples were collected after primary infection in septic patients. Control samples were collected from matched healthy blood donors (age ± 10 years, sex, race).
Circulating PBMC and monocytes were isolated with Human monocytes separation medium kit (Sangon Biotech, China), and monocytes were harvested by removing non-adhesion cells. After washing with PBS several times, RNA of monocytes and PBMC was extracted, followed with Real-time PCR.