Yeast two-hybrid screening
The yeast two-hybrid experiment was performed by Jun Yang following the protocol as previously described [53] in Dr. J. Kevin Foskett’s laboratory at the University of Pennsylvania, Perelman School of Medicine. Briefly, a cDNA fragment encoding the carboxyl terminus of rat InsP3R1 aa 2590-C was cloned into the pLexA vector (Clontech) to be used as a bait to screen a human fetal brain cDNA library (Invitrogen). Library screening was performed following the company’s instructions. From 6 × 106 primary transformants we discovered 14 positive colonies. The library plasmids were recovered using a yeast plasmid extraction kit (Qiagen). The prey sequences were identified by the DNA sequence facility of the University of Pennsylvania. Positive plasmids were confirmed by co-transformation of the library and bait plasmids for comparison with non-specific interactions by co-transformation of the library plasmid and the empty pLexA vector.
Cell culture
Murine macrophage RAW264.7 and human embryonic kidney (HEK) 293 cells were purchased from ATCC, and grown in Dulbecco’s modified Eagle’s medium (DMEM) and minimum essential medium containing 10% fetal bovine serum (FBS), respectively. Chicken B-lymphoid cell line DT40 cells were derived from Dr. J. Kevin Foskett’s laboratory. They were maintained in RPMI 1640 containing 10% FBS, 1% chicken serum, 1% Antibiotic-Antimycotic at 37°C, 95% humidity, 5% CO2.
Mouse macrophage differentiation and culture
Primary cultures of BMDMs from C57BL/6 mice were prepared as previously described [54]. In brief, bone marrow cells were aseptically collected from the female mice aged 6–8 weeks by flushing the euthanized mice’s leg bones with phosphate-buffered saline (PBS). The cells were then incubated in red cell lysis buffer (155 mM NH4Cl, 10 mM NaHCO3, and 0.1 mM ethylenediaminetetraacetic acid (EDTA)) before centrifuging. The cells were cultured for 7 days in DMEM containing 20% FBS, 100 μg/ml streptomycin, 55 mM mercaptoethanol, 100 U/ml penicillin, and 30% conditioned media from L929 cells expressing macrophage colony-stimulating factor (M-CSF). Non-adherent cells were removed. Flow cytometry analysis indicated that the harvested cell population contained 86–95% CD11b + F4/80 + cells.
Plasmid construction
Human full-length SEC5 cDNA and IRF-3 cDNA were purchased from YR Gene. Plasmids were constructed according to standard protocols, i.e., by cloning the cDNA corresponding to the residues of interest into vectors including pET28a, pCDNA3.1-V5-His (Life Tech), pECFP-N1 (Clontech), and pGEX-6P-1 (GE). Restriction enzymes for cloning were purchased from New England Biolabs or Takara.
C. albicans infection model and fungal burden
For in vivo C. albicans infection, a group of C57B/L6 female mice aged 6–8 weeks were injected via lateral tail vein with 1 × 105 CFUs of C. albicans in 200 μl sterile saline. One hour later, the mice were injected i.p. with H1-Tat peptide or H1-scrambled-TAT peptide at 2 mg/kg, or with SEC5-2 peptide at 50 μg/kg. Kidney CFU assays were performed 2 days after infection. The kidneys were removed, weighed, and homogenized in sterile PBS in a tissue grinder. The number of viable Candida cells in the tissues was determined by plating serial dilutions on SD agar plates. The CFUs were counted after 24 h of incubation at 30°C and expressed as CFUs per gram of tissue.
Recombinant protein purification from E. coli and GST pull-down assays
Glutathione S-transferase (GST) fusion proteins were expressed in BL21 (DE3)pLysS E. coli and purified with Glutathione Sepharose 4B following the manufacturer’s protocol (GE). His-tagged proteins were expressed in BL21 (DE3)pLysS E. coli using pET28A vectors and purified using Ni-columns according to the manufacturer’s protocol (Qiagen). Purified proteins were dialyzed against 1 × Dulbecco’s PBS (DPBS) buffer. Unless specified, pull-down assays and western blotting were performed according to standard protocols. Cellular lysates, prepared using 1 × DPBS containing 1% Triton X-100, 0.5–1 mg/ml total cellular protein (dependent on expression), in a total volume of 1 ml, were mixed with 20 μg GST fusion protein bound to beads and incubated at 4°C while rotating for 2 h. The beads were spun down and washed thrice using lysate buffer. Bound proteins were eluted using 1 × loading buffer and detected by western blotting.
Co-immunoprecipitation
RAW264.7 cells, stimulated with C. albicans (multiplicity of infection (MOI) = 10) or not, were lysed using 1 × DPBS containing 1% Triton X-100. Ten micrograms of rabbit InsP3R1 or InsP3R3 antibodies, or an equivalent amount of rabbit immunoglobulin G (IgG) isotype control (Sigma; #18140), was conjugated with 30 μl of a 50% slurry of protein G agarose resin according to the manufacturer’s instructions (Yeasen; #36405ES08). Then, the antibody-conjugated agarose resin was incubated with protein lysates (500 mg of protein) with gentle agitation for 2 h. The beads were retained after three washes with PBS. Next, 1 × loading buffer was added to dissociate immunoprecipitates, and western blotting was performed accordingly.
Acceptor photobleaching fluorescence resonance energy transfer (FRET)
Acceptor photobleaching FRET was measured by confocal microscopy (A1R, Nikon, Japan) according to a previously described method [29, 30]. Energy transfer was detected as an increase in donor fluorescence (Alexa Fluor 488) following the complete photobleaching of the Cy3 acceptor molecules. The amount of energy transfer at co-localization areas was calculated as the percentage increase in donor fluorescence, and non-co-localization areas were selected as negative controls. The imaging sequence was as follows: First, donor and acceptor channels were sequentially scanned at low laser intensity before the acceptor was bleached with the 561-nm laser line at 70% intensity, and the donor and acceptor channels were scanned again at low laser intensity. Acquired images were exported to 8-bit TIFF format, and FRET efficiency was analyzed using Nikon NIS-Elements software.
Immunofluorescence staining
BMDM cells at 7 × 105 were placed on 12-mm coverslips (Fisher Scientific, UK) and incubated with C. albicans at 37°C in 5% CO2 for 60 min. The cells were fixed using 4% paraformaldehyde (PFA) in DPBS, followed by blocking and permeabilization with 0.1% IGEPAL (Sigma, St. Louis, MO, USA) in DPBS with 2% bovine serum albumin (BSA) (Amresco, Solon, OH, USA). Primary antibodies, diluted in 2% BSA, were applied at 4°C overnight. The cells were subsequently washed four times with DPBS before being incubated with the appropriate secondary antibodies (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) diluted in 2% DPBS. The coverslips were washed four times with DPBS before being mounted using Vectashield. Confocal images were acquired with a Nikon A1R confocal system. Pearson’s correlation coefficients were calculated using the NIS-Elements AR 4.5 software to show the overlap between Alexa Fluor 488 and Cy3 at each co-localization area.
Calcium imaging
HEK293 cells were seeded in 35-mm dishes and incubated with 2 μM Fura-2 AM (Invitrogen, Thermo Fisher Scientific, MA, USA) in Hank’s balanced salt solution (HBSS, Sigma, St. Louis, MO, USA) containing 0.04% Pluronic F-127 (Sigma) for 30 min, in normal culture media, at 37°C and 5% CO2. The cells were then washed and continuously perfused with HBSS containing 1.8 mM CaCl2 and 0.8 mM MgCl2, pH 7.4. In the experiments to measure calcium imaging, the cells were perfused with HBSS containing 50 μM carbachol. Fura-2 was alternately excited at 340 and 380 nm, and the emitted fluorescence, filtered at 510 nm, was recorded using Nikon NIS-Elements software. Individual cells were chosen as different regions of interest (ROIs), and the ratio (340/380) of each ROI in all time frames was obtained after background correction. Dye calibration was performed, and ratios were then converted to cytosolic Ca2+ concentrations by the Grynkiewicz equation: [Ca2+] = Kd*β*(R-Rmin)/(Rmax-R) [55].
Nuclear patch-clamp recording of InsP3R
Nuclear patch-clamp recording was performed as previously described [35,36,37]. Briefly, DT40TKO-InsP3R3 nuclei were isolated by homogenizing cells in nuclei isolation solution. Then, 80 μl of cell homogenate was added to 2 ml of bath solution. Single InsP3R channels were detected using a HEKA EPC-10 amplifier (HEKA Elektronik) and pipettes filled with pipette solution. Peptides were directly added to the patch pipette solution. Single-channel analysis was performed using QuB software (University of Buffalo).
Transfection with siRNA and plasmids
The cells were seeded in 6-well plates (1 × 106 cells/well) in 1 ml DMEM containing 10% FBS before being transfected with 120 pmol siRNA against SEC5, non-targeting control siRNA, or 2 μg SEC5-EGFP or pcDNA-EGFP control plasmids, using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s protocol. The cells were lysed 48 h after transfection, and the relative intensities of SEC5 protein were determined by western blotting.
Phagocytosis assays
Phagocytosis assays were performed using the standard protocol with modifications [38]. Briefly, C. albicans (SC5314) cells were added to 1 × 106 BMDM macrophages in glass-based imaging dishes at an MOI of 5. Video microscopy was performed using a Nikon ECLIPSE Ti microscope in a 37°C chamber, and images were captured at 20-ss intervals over a 1-h period using an EMCCD camera. Live-cell video microscopy enables internalization of C. albicans to be visualized without the need to stain external Candida. At least three independent experiments were performed for each group and at least three videos were analyzed from each experiment using ImageJ analysis software.
In vitro TBK1 kinase assays
TBK1 kinase assays were performed using the standard protocol with modifications [56]. Endogenous TBK1 from COS-1 cells was added to each kinase activity assay in a buffer containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.6), 0.1 mM sodium vanadate, 20 mM β-glycerophosphate, 10 mM MgCl2, 50 mM NaCl, and 100 μM ATP. For each reaction, 20 μg of purified recombinant GST-tagged IRF-3-C was used as a substrate for TBK1, and the reactions were carried out for 30 min at 30°C. The reactions were terminated by adding sample buffer. The phosphorylation of recombinant GST-IRF3-C was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with phosphor-IRF-3 (Ser396) antibodies (Cell Signaling Technology, Cat#29047), and the equivalent protein concentrations were confirmed by immunoblotting with anti-TBK1 and anti-GST antibodies.
Cytokine measurements
The enzyme-linked immunosorbent assay kits for IFN-β were purchased from NeoBioscience. All samples were measured in triplicate according to the manufacturer’s protocol.
Statistical analysis
At least three biological replicates were performed for all experiments, unless otherwise indicated. Individual data for each experiment are provided as Additional file 11. Student’s t test was used for statistical analyses of paired observations. Differences between means were accepted as statistically significant at the 95% level (p < 0.05).
Antibodies
The primary antibodies used for immunoblotting were rabbit monoclonal anti-TBK1 (Cell Signaling Technology, Danvers, MA, USA, Cat# 3504S RRID:AB_225566,1:1000 dilution), rabbit monoclonal anti-phosphorylated TBK1 (phosphor S172) (Cell Signaling Technology Cat# 5483P RRID:AB_10693472,1:1000), rabbit monoclonal anti-β-actin (Cell Signaling Technology Cat# 4970S RRID:AB_2223172, 1:1000), rabbit monoclonal anti-PCNA (Cell Signaling Technology Cat# 13110 RRID:AB_2636979,1:1000), rabbit monoclonal anti-IRF3 (phosphor Ser396) (Cell Signaling Technology Cat# 4302S RRID:AB_1904036,1:1000), rabbit monoclonal anti-phosphorylated IRF-3 (Cell Signaling Technology Cat# 29047 RRID:AB_823547,1:1000), rabbit polyclonal anti-SEC5 (Sigma-Aldrich, Atlas Antibodies Cat# HPA032093 RRID:AB_10611964, 1:1000), mouse monoclonal anti-InsP3R-3 (BD Biosciences, Franklin, NJ, USA,BD Biosciences Cat# 610313 RRID:AB_397705,1:1000), rabbit polyclonal anti-GST (ABclonal Technology, Wuhan, China, ABclonal Technology Cat#AE006,1:1000), and mouse monoclonal anti-His tag (ABclonal Technology Cat#AE003,1:1000).
The antibodies used for immunofluorescence were rabbit monoclonal anti-TBK1 (Abcam, Cambridge, UK, Abcam Cat# ab40676 RRID:AB_776632,1:100 dilution), mouse monoclonal anti-InsP3R-3 (BD Biosciences Cat# 610313 RRID:AB_397705, 1:100), rabbit polyclonal anti-SEC5 (Atlas Antibodies Cat# HPA032093 RRID:AB_10611964, 1:100), Alexa Fluor 488-conjugated donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (Life Technologies, Molecular Probes Cat# A-21202 RRID:AB_141607,1:400), and Cy3-conjugate donkey anti-rabbit IgG, species adsorbed, antibody (Millipore, Burlington, MA, USA, Millipore Cat# AP182C RRID:AB_92588,1:400).
Polyclonal antibody anti-Type 1 InsP3R was generated by Abgent Biotech. Co. Ltd. (Shanghai, China) against the peptides RIGLLGHPPHMNVNPQQPA, strictly following Akihiko Tanimura’s method [57]. Antigens were prepared by GL Biochem. Ltd. (Shanghai, China). The specificity of the InsP3R1 antibody has been previously validated [57, 58]. We also reconfirmed the specificity of the polyclonal antisera made against the peptides from the type 1 InsP3 receptors in chicken DT40 cells with all InsP3R isoforms genetically deleted (DT40-KO) and engineered to stably express InsP3R type 1 (KO-R1) or type 2 (KO-R2), which were provided by Dr. J. Kevin Foskett. The mouse monoclonal antibody against InsP3R3 was purchased from BD Biosciences. Its specificity was confirmed by western blot of DT40-KO stably expressing a mutant InsP3R3, which agreed with the results of immunocytochemistry and western blotting assay of InsP3R3 by other groups [58, 59].
Reagents
Inositol-1,4,5-trisphosphate (Cat#10008205) and araguspongin B (Cat#10006797) were from Cayman Chemical (Ann Arbor, MI, USA). The other reagents are indicated as follows:
5’-GGUCGGAAAGACAAGGCAGTT-3’
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SiRNA-Negative Control (Shanghai GenePharma Co., Ltd., Shanghai, China)
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5’-UUCUCCGAACGUGUC ACG UTT-3’
ShRNA-SEC5 (Shanghai Genechem Co., Ltd., Shanghai, China)
5’-CCGGGCCGAAGAGATAAAGAGATTACTCGAGTAATCTCTTTATCTCTTCGGCTTTTTT-3’
Animals
C57BL/6 mice were purchased from Shanghai BK Animal Model Inc. Ltd., China.