Human PBMC learn about
Entire blood samples have been amassed from each and every learn about player. Human PBMCs have been remoted with Ficoll-PaqueTM PREMIUM (17-5442-02, GE Healthcare) from freshly amassed peripheral venous blood from wholesome controls in line with the producer’s directions. In brief, Ficoll-Paque medium used to be added to the centrifuge tube, and the diluted blood pattern used to be layered onto the Ficoll-Paque medium resolution. The tube used to be centrifuged at 400 × g for 30–40 min at 18–20 °C with the brake became off. The higher layer containing plasma and platelets used to be aspirated the usage of a sterile pipette, leaving the layer of mononuclear cells undisturbed on the interface. The layer of mononuclear cells used to be washed two times with 1× PBS.
For the siRNA knockdown assays, human PBMCs have been transfected with RfectSP siRNA Transfection Reagent (BioGenerator Biotechnology) in line with the producer’s directions. 5 days after transfection, human PBMCs have been stimulated with HKCA (MOI = 2) or α-mannan (100 μg/mL) and harvested for gene expression research.
For stimulation of human PBMCs, cells have been stimulated with curdlan (100 μg/mL), HKCA (MOI = 2), or α-mannan (100 μg/mL). Cells have been then harvested for gene expression research. This learn about adopted the tips established by way of the Declaration of Helsinki, and the protocol used to be licensed by way of the Ethics Committee of Tongji Clinical Faculty, Huazhong College of Science and Generation (venture quantity: S309). All learn about individuals signed a written knowledgeable consent shape.
Dock2 gene KO mice have been a type present from Professor Chaohong Liu at Tongji Clinical Faculty of Huazhong College of Science & Generation and Professor Yoshinori Fukui at Kyushu College . Mice have been housed below SPF prerequisites, and the experimental protocols have been licensed by way of the Institutional Animal Care and Use Committee of Huazhong College of Science and Generation (venture quantity: S909). This learn about complied with all related moral laws for animal analysis.
293T and HeLa cells have been maintained in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin. THP-1 cells have been maintained in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin–streptomycin and 1% sodium pyruvate.
Preparation of mouse BMDMs
BMDMs have been received by way of differentiating bone marrow progenitors from the tibiae and femora of 6–8-week-old male or feminine mice in Iscove’s changed Dulbecco’s medium containing 20 ng/mL M-CSF, 10% heat-inactivated FBS (Invitrogen), 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen) for five–7 days. Cells have been then replated in 6-well or 12-well plates 1 day sooner than experiments.
The anti-p-SYK (clone C87C1), anti-p-IκBα (clone 14D4), anti-p-JNK (clone 81E11), anti-HA (clone C29F4) and anti-Flag (clone D6W5B) antibodies have been bought from Cellular Signaling Generation (cat nos. 2710, 2859, 4668, 3724 and 14793, respectively). The anti-HA (clone HA-7) and anti-Flag (clone M2) antibodies used for immunoprecipitation have been bought from Sigma (cat nos. H9658 and F1804, respectively). The anti-MYC antibody used to be bought from GNI (GNI4110-MC). The anti-DOCK2 (clone E-7), anti-p-ERK (clone 12D4), anti-p-tyrosine (clone PY20), anti-HSP90 (clone AC-16) and anti-Actin (clone C-2) antibodies have been bought from Santa Cruz Biotechnology (cat nos. sc-365242, sc-81492, sc-508, sc-130068 and sc-8432, respectively). The anti-SYK, anti-ERK1/2, anti-JNK1, anti-GAPDH, and anti-FcRγ antibodies have been bought from ABclonal (cat nos. A2123, A4782, A0288, A19056, and A12889, respectively). The anti-RAC1 and anti-RAC2 antibodies have been bought from Proteintech (cat nos. 24072-1-AP and 60077-1-Ig, respectively). The anti-CD45 (clone 30-F11), anti-Ly6G (clone 1A8), anti-F4/80 (clone BM8) and anti-CD11b (clone M1/70) antibodies used for waft cytometry have been bought from Biolegend (cat nos. 103108, 127608, 123115, and 101212, respectively). A Zombie Violet Fixable Viability Package used to be bought from Biolegend (cat no. 423114). Curdlan used to be bought from InvivoGen (cat no. tlrl-cud). α-Mannan used to be bought from Sigma (cat no. M3640). NSC23766 and NAC have been bought from Topscience (cat nos. T6342 and TP1088, respectively). fMLP used to be bought from MCE (HY-P0224). Recombinant mouse M-CSF protein used to be bought from Peprotech (cat no. 315-02). Mouse CXCL1 and TNF-α ELISA kits have been bought from Biolegend (447507 and 430904, respectively). A DNA purification package used to be received from Axygen. Inorganic pyrophosphatase, rNTPs, DNase I, E. coli PolyA Polymerase, Vaccinia capping enzyme and a Monarch RNA Cleanup Package have been received from New England Biolabs. The reversed-phase column used to be received from Agilent. Dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dimyristoyl-rac-glycero-3-methoxypoly(ethylene glycol)-2000 (DMG-PEG2000) have been equipped by way of A.V.T. (Shanghai) Pharmaceutical Co., Ltd. Ldl cholesterol used to be received from Aladdin (Shanghai, China).
Cells have been fastened with 4% paraformaldehyde and have been then permeabilized with PBS containing 0.3% Triton X-100 for 10 min. Previous to incubation with the main antibody, the samples have been incubated with 10% goat serum at room temperature for 1 h to dam nonspecific staining. After 12 h of incubation with the main antibody at 4 °C, the samples have been washed thrice with ice-cold PBS and extra stained with fluorophore (Alexa 488 and Alexa 594)-conjugated secondary antibodies. After antibody staining, the samples have been counterstained with DAPI and immersed in mounting medium sooner than being sealed on a slide with nail polish. Sealed slides have been analyzed the usage of an Olympus FV3000 microscope with the spouse device.
Immunoblotting and immunoprecipitation
Cells have been harvested and lysed on ice in lysis buffer containing 0.5% Triton X-100, 20 mM HEPES (pH 7.4), 150 mM NaCl, 12.5 mM β-glycerophosphate, 1.5 mM MgCl2, 10 mM NaF, 2 mM dithiothreitol, 1 mM sodium orthovanadate, 2 mM EGTA, 20 mM aprotinin, and 1 mM phenylmethylsulfonyl fluoride for 30 min and have been then centrifuged at 12,000 rpm for 15 min to acquire clarified lysates. For immunoprecipitation, cellular lysates have been incubated with 1 μg of antibody at 4 °C in a single day previous to incubation with protein A-Sepharose or protein G-Sepharose beads for two h. The beads have been washed 4 occasions with lysis buffer, and the precipitates have been eluted with 2× pattern buffer and immunoblotted with antibodies.
Lentivirus-mediated gene knockout in THP-1 cells
The pLentiCRISPR-GFP vector used to be used for CRISPR/Cas9-mediated gene KO within the THP-1 cellular line. In brief, a lentiviral vector expressing a gRNA used to be transfected along side packaging plasmids into HEK293T (ATCC) packaging cells. Then, 48 and 72 h after transfection, virus-containing supernatants have been harvested and filtered via a nil.2 μm clear out. Goal cells have been inflamed two times and looked after by way of waft cytometry. For some experiments, unmarried cells have been looked after into 96-well plates by way of waft cytometry for isolation of unmarried clones. Remoted unmarried clones have been verified by way of western blot research and DNA sequencing.
Era of strong cellular traces
For lentivirus packaging, 6.0 μg of the pCDH-MSCV-MCS-EF1-copGFP-T2A-Puro plasmid harboring DOCK2-Flag or DOCK2-Y985 + 1405F-Flag, 3.0 μg of the pMD2.G plasmid, and three.0 μg of the psPAX2 plasmid have been cotransfected into HEK293T cells in a ten cm tradition plate (8 × 106 cells according to plate). Twelve hours publish transfection, the medium used to be modified to recent DMEM supplemented with 10% FBS, 5 mg/mL penicillin and 10 mg/mL streptomycin. 40-eight hours publish transfection, supernatants containing lentiviral debris have been amassed, 8 mL of each and every supernatant used to be used for 2 rounds of an infection of DOCK2−/− THP-1 cells (3 × 106 cells according to plate). Inflamed cells have been decided on with a last puromycin focus of 6 µg/mL for 72 h and have been then amassed by way of centrifugation (1000 × g, 5 min) and cultured. The certain cellular traces have been recognized by way of immunoblotting with an anti-Flag antibody.
RT and Actual-time PCR
General RNA used to be extracted from the spinal twine with TRIzol in line with the producer’s directions. One microgram of general RNA for each and every pattern used to be opposite transcribed the usage of SuperScript® II Opposite Transcriptase from Thermo Fisher Clinical. The ensuing complementary DNA used to be analyzed by way of real-time PCR the usage of SYBR Inexperienced Actual-Time PCR Grasp Combine. All gene expression effects are expressed as arbitrary devices relative to the expression of Actb or GAPDH.
Go with the flow cytometry
Someday after an infection with reside C. albicans, mice have been sacrificed and perfused with 1× PBS. Kidneys have been homogenized in ice-cold tissue grinders and filtered via a 100 μm cellular strainer, and the cells have been amassed by way of centrifugation at 400 × g for five min at 4 °C. Cellular floor staining used to be carried out for 30 min at 4 °C. Zombie Violet Fixable Viability reagent (1:400; Biolegend) used to be added to exclude useless cells. Go with the flow cytometry knowledge research used to be carried out by way of the usage of CytExpert.
RAC1 task analysis
A complete of 8 × 106 WT THP-1 cells or DOCK2-KO THP-1 cells have been seeded in 10-cm plates. After in a single day serum hunger, cells have been inflamed for 0, 15, and 30 min with α-Mannan (100 μg/mL) or HKCA (MOI = 2). Cells have been washed two times with ice-cold 1× PBS and lysed in lysis buffer containing 50 mM Tris (pH 7.5), 10 mM MgCl2, 0.5 M NaCl, and a pair of% IGEPAL, as described within the Rac1/Cdc42 Activation Assay Package handbook (Upstate). The supernatants have been incubated with GST-PBD-agarose beads at 4 °C for three h, washed thrice in wash buffer (25 mM Tris (pH 7.5), 30 mM MgCl2, 40 mM NaCl) and resuspended in loading buffer for immunoblot research.
ROS manufacturing assay
For the ROS manufacturing assay, ROS ranges have been measured with a DHE (dihydroethidium) fluorescent probe in line with the producer’s directions. In brief, 2 × 105 WT THP-1 cells or DOCK2-KO THP-1 cells have been differentiated with PMA for two days and have been then washed two times with 1 × PBS. DHE used to be added to recent medium at a last focus of one μM, and the cells have been incubated at 37 °C for 30 min sooner than stimulation with Curdlan (100 μg/mL), Mannan (100 μg/mL) or HKCA (MOI = 2). ROS manufacturing used to be measured by way of waft cytometry.
Mass spectrometry identity of DOCK2 phosphorylation websites
For identity of the websites in DOCK2 phosphorylated by way of SYK, 293T cells have been transfected with Flag-hDOCK2 and empty vector or Flag-hDOCK2 plus HA-hSYK and have been then subjected to immunoprecipitation with an anti-Flag antibody 2 days after transfection. After washing 4 occasions with IP buffer, proteins have been eluted with IP buffer containing 0.1% SDS and analyzed by way of mass spectrometry. Samples have been diminished and alkylated in dithiothreitol (DTT) and iodoacetamide previous to trypsin digestion in a single day. Digested samples have been injected onto an Agilent Zorbax 300SB-C18 0.075 mm × 150 mm column in an Eksigent NanoLC device coupled to a Thermo LTQ-ETD Orbitrap mass spectrometer. An Advion TriVersa NanoMate used to be used because the nano-electrospray ionization supply, and MS knowledge have been searched in opposition to the RefSeq human protein database with Sorcerer Sequest. The searched dataset used to be processed throughout the TPP (Trans-Proteomics Pipeline) and filtered with Peptide Prophet.
In vitro fungal killing assay
For the in vitro fungal killing assay, differentiated WT THP-1 cells or DOCK2-KO THP-1 cells (5 × 105 cells/properly) have been incubated with reside C. albicans (MOI = 1) for twenty-four h. After coculture, cells have been washed with 1 × PBS thrice and have been then resuspended in recent medium containing amphotericin B (Sigma–Aldrich, V900919) at a last focus of 30 μg/mL and cultured for three h at 37 °C. Cells have been washed once more thrice in 1× PBS and have been then lysed by way of the addition of 0.02% Triton X-100 (Sigma–Aldrich, T8787), and 100 mL of the suspension (1:1000 dilution) used to be unfold on YPD plates. After incubation at 37 °C for twenty-four h, killing used to be evaluated by way of counting the Candida colonies.
Membrane and cytoplasmic fractionation
WT THP-1 cells and DOCK2-KO THP-1 cells have been rinsed with bloodless 1× PBS thrice and washed one time in hypotonic buffer (10 mM KCl, 1.5 mM MgCl2, and 10 mM Tris-HCl (pH 7.5)) supplemented with a protease inhibitor, incubated on ice in hypotonic buffer for 15 min after which pipetted up and down 5–10 occasions. The lysates have been centrifuged at 4 °C for five min at 2500 × g to take away nuclei and mobile particles. Supernatants have been centrifuged at 100,000 × g for 60 min at 4 °C to split the cytosolic extracts (S100) and pellets (P100). The pellets (P100) have been resuspended in lysis buffer volumes equivalent to these of the supernatants (S100), saved with the addition of five× loading buffer, and analyzed by way of western blotting.
Synthesis of single-stranded mRac1 mRNA
In vitro transcription (IVT) the usage of VSW-3 RNA polymerase  used to be performed to organize mRac1 mRNA. DNA fragments containing the 5′UTR series, Flag coding series, mRac1 coding series and three′UTR series below the keep watch over of the VSW-3 RNA polymerase promoter  have been inserted into the HhaI and XbaI websites within the pUC18 plasmid. Then, a couple of amplification primers (F-primer: 5′-CGTTGTAAAACGACGGCCAGTGC-3′ and R-primer: 5′- ATAAAATTAAGTTGCATCAAGCT-3′) have been used for PCR amplification of the DNA template for IVT. PCR merchandise have been wiped clean with a DNA purification package. The IVT response (200 μL) contained 40 mM Tris-HCl (pH 8.0), 16 mM MgCl2, 5 mM DTT, 2 mM spermidine, 35 ng/μL template DNA, 1.5 U/μL RNase inhibitor, 0.2 μM inorganic pyrophosphatase, each and every of the 4 NTPs (4 mM) and nil.15 μM VSW-3 RNA polymerase. After incubation at 25 °C for 12 h, 40 devices of DNase I have been added to the response aggregate and incubated for an extra 30 min at 37 °C to take away the template DNA. RNA merchandise have been purified with a Monarch® RNA Cleanup package in a last quantity of 200 μl. Linear RNA used to be capped (cap0) the usage of Vaccinia capping enzyme (NEB) in line with the producer’s directions. Polyadenosine tails have been added to the capped linear transcripts the usage of E. coli PolyA Polymerase (NEB) in line with the producer’s directions, and entirely processed mRNA used to be purified with a Monarch® RNA Cleanup Package. mRNA used to be additional purified via a 25 × 150 mm reversed-phase column with a particle measurement of 10 μm and pore measurement of 4000 A° (Agilent; section quantity: PL1212-3103) on an Agilent 1260 Sequence HPLC device (Agilent). The precise sequences of the mRNAs and the corresponding translated proteins are equipped in Desk S1.
Manufacturing of lipid-based mRac1 nanoparticles (mRac1@LNPs)
mRNA-loaded LNPs have been produced by way of the ethanol dilution means. All lipids, particularly, DLin-MC3-DMA, ldl cholesterol, DSPC, and DMG-PEG2000, have been dissolved in ethanol at a molar ratio of fifty:10:38.5:1.5. mRac-1 diluted in 10 mM sodium citrate buffer (pH 4.5) used to be all of a sudden combined with the answer of the above lipids at a quantity ratio of three:1 (aqueous:natural) by way of pipetting and vortexing. After incubation for 20 min at room temperature, the ensuing nanoparticles (mRac1@LNPs) have been diluted with 1× PBS (pH 7.4) buffer for characterization and mRNA transfection in vitro. For mRac-1 transfection in vivo, the freshly produced nanoparticles have been dialyzed in opposition to 1× PBS to fully take away the ethanol.
Characterization of mRac1@LNPs
mRNA encapsulation by way of LNPs used to be evaluated by way of a gel retardation assay. The mRac1@LNPs have been formulated at quite a lot of weight ratios of general lipids to mRNA and have been then electrophoresed on a 2% agarose gel in 1× TAE buffer at 100 V for 10 min. The mRNA used to be stained with Gel Purple (Biosharp, China) and visualized with a ChemiDoc XRS+ imaging device (Bio–Rad, USA).
The particle measurement and zeta possible of mRac1@LNPs have been analyzed the usage of a Zetasizer Nano ZS90 tool (Malvern, UK) after dilution with 1 × PBS. To look at the morphology of mRac1@LNPs, the mRac1@LNPs resolution used to be deposited onto carbon TEM grids and imaged by way of TEM (HT7700, Hitachi, Japan).
The stableness of mRac1@LNPs in vitro and in vivo used to be evaluated by way of measurement and PDI dimension. The particle measurement and PDI of mRac1@LNPs after incubation with 10% FBS at 37 °C or garage at 4 °C in PBS have been analyzed as up to now described. To check the biosafety of mRac1@LNPs in vivo, a hemolysis assay used to be performed by way of incubation with crimson blood cells remoted from mice. The relative hemolysis of mRac1@LNPs or clean LNPs used to be analyzed in accordance with standard saline because the damaging keep watch over (0%) and 1% Triton X-100 because the certain keep watch over (100%).
For histopathological analyses, kidneys have been fastened with 10% neutral-buffered formalin, processed in line with same old procedures, embedded in paraffin, and sectioned. Two-micrometer-thick sections have been stained with hematoxylin and eosin (H&E) and PAS. Renal irritation used to be scored in accordance with the H&E and PAS staining proportion (proportion of the renal parenchyma and/or pelvis concerned with tubulointerstitial nephritis and/or pyelonephritis) as now not vital (rating 0), lower than 10% (rating 1), 10–25% (rating 2), 25–50% (rating 3), or more than 50% (rating 4). The intralesional fungal burden used to be scored in accordance with PAS staining as now not vital (rating 0); scant presence, in lower than 10% of inflammatory foci (rating 1); gentle to average presence, in 10–25% of inflammatory foci (rating 2); average to vital presence, in 25–50% of inflammatory foci (rating 3); or vital presence, in additional than 50% of inflammatory foci (rating 4).
Systemic C. albicans an infection style
Are living cells of C. albicans pressure SC 5314 (4 × 105 yeast cells in 0.1 mL of one× PBS buffer) have been injected intravenously into 6- to 8-week-old littermate mice of various genotypes. Inflamed mice have been monitored day by day for weight reduction and survival. The fungal burden within the kidneys used to be measured 2 days after an infection. After the kidneys have been amassed, tissue homogenates have been serially diluted and plated on yeast extract–peptone–dextrose agar. Fungal colony-forming devices have been counted 24 h after plating. For the in vivo mRNA@LNP remedy experiment, feminine WT mice have been intravenously injected with reside C. albicans (4 × 105 cfu/100 μL 1 × PBS), and 12 h later, the mice have been intravenously injected with keep watch over GFP@LNPs (0.25 μg/g) or mRac1@LNPs (0.25 μg/g). For the fMLP remedy experiments, WT mice have been intravenously injected with reside C. albicans (4 × 105 cfu/100 μL 1 × PBS), and 12 h later, the mice have been injected intraperitoneally with DMSO (50 μL) or fMLP (0.5 μg/g/50 μL).
Human PBMCs learn about
Entire blood samples have been amassed from wholesome individuals. Human PBMCs have been remoted with Ficoll-PaqueTM PREMIUM (17-5442-02, GE Healthcare) from freshly amassed peripheral venous blood from wholesome donors. In brief, Ficoll-Paque medium used to be added to the centrifuge tube, and the diluted blood pattern used to be layered onto the Ficoll-Paque medium resolution. The tube used to be centrifuged at 400 × g for 30 to 40 min at 18 °C to twenty °C with the brake became off. The higher layer containing plasma and platelets used to be aspirated the usage of a sterile pipette, leaving the layer of mononuclear cells undisturbed on the interface. The layer of mononuclear cells used to be washed two times with 1× PBS. For the siRNA knockdown assays, human PBMCs have been transfected with RfectSP siRNA Transfection Reagent (BioGenerator Biotechnology) in line with the producer’s directions. 5 days after transfection, human PBMCs have been stimulated with Curdlan (100 μg/mL), HKCA (MOI = 2) or α-mannan (100 μg/mL) and harvested for gene expression research. This learn about adopted the tips established by way of the Declaration of Helsinki, and the protocol used to be licensed by way of the Ethics Committee of Tongji Clinical Faculty, Huazhong College of Science and Generation. All learn about individuals signed a written knowledgeable consent shape.
The importance of variations between two teams used to be made up our minds by way of unpaired two-tailed t check, multiple-group comparisons have been carried out the usage of one-way ANOVA, the medical ratings and weight curves have been analyzed by way of two-way ANOVA with a number of comparisons, and the survival charge used to be analyzed by way of the usage of the log-rank (Mantel–Cox) check. P < 0.05 used to be regarded as to signify importance. The consequences are proven because the approach, and the mistake bars point out the usual error of the imply (S.E.M) of organic or technical replicates as indicated within the determine legend.