Chemicals
Hyclone SFX-insect cell culture medium was purchased from Thermo Scientific (USA), and fetal bovine serum (FBS) was purchased from Gibco (USA). IPG drystrip and IPG buffer were purchased from Amersham Biosciences (Uppsala, Sweden). Azadirachtin (95%) was obtained from Sigma (St.Louis, MO, USA). Other chemicals were domestic products with analytical grade. Rabbit polyclonal antibodies against HSP40, TCTP and GAPDH, respectively, were obtained from BOSTER (Wuhan, China).
Cell culture
Sf9 cells were obtained from the State Key Laboratory for Biocontrol/Institute of Entomology, Sun Yat-Sen University (Guangzhou, China), and were maintained at 27 °C in 25 cm2 culture flasks (Corning, USA) containing 3 mL Hyclone SFX-insect cell culture medium supplemented with 5% fetal bovine serum. The doubling time under optimum conditions was 18–24 h and cells were subcultured every 2 days.
Cell viability assay
Sf9 cells were seeded onto a 96-well plate (5 × 103/well) and incubated for 24 h, then exposed to a series of concentrations of azadirachtin for 24 h and 0.1% DMSO was included as a control. Fifty μL of methylthiazoletetrazolium (MTT) solution was added to each well and cells were incubated in dark for another 4 h. After removing the supernatant, 150 μL DMSO was added and mixed thoroughly with the pipette. Cell viability was measured based on absorbance at 490 nm using a microplate reader (Thermo Scientific, Waltham, MA, USA).
Morphological observation by inverted phase contrast microscopy
Cells seeded in 6-well plates were treated with 0.75 μg/mL azadirachtin and 0.1% DMSO was used as control. Morphological characteristics of cells at 0, 24, 48, and 72 h after treatments were recorded by an inverted phase contrast microscope (Lecia, Japan), respectively.
Hoechst 33258 analysis
Hoechst 33258 is a blue fluorescent dye that could penetrate cell membranes and stain the cell nuclei with blue color. Sf9 cells treated with azadirachtin for 24, 36 and 48 h were stained with 0.5 mL Hoechst 33258 solution for 5 min and then washed with phosphate-buffered saline (PBS) twice for 3 min each time. The stained cells were observed under the fluorescent microscope (Nikon, Japan).
Analysis of caspase-3-like enzymatic activity
Sf9 cells treated with azadirachtin were collected and the caspase-3-like proteolytic activity was measured using a Caspase-3 Colorimetric Assay Kit (KeyGEN BioTECH, Nanjing, China). The cells were then washed with PBS twice and collected by centrifugation at 2000 rpm for 5 min. Total cellular proteins were extracted using a cold lysis buffer on ice for 20–60 min. Protein concentrations were determined following the Bradford approach [24]. The solution containing 150 mg proteins with the Caspase-3 substrate (integrating specific luminescence substrate) was incubated in dark at 37 °C for 4 h. Caspase-3-like enzymatic activity was measured based on the absorbance of samples measured at 405 nm using a microplate reader (Thermo Scientific, USA). The Cacpase-3 inhibitor Z-VAD-FMK was also used with the final concentration of 20 μM.
Preparation of protein samples
The adherent Sf9 cells treated with 0.75 μg/mL azadirachtin for 24 h and control group were washed with PBS twice and then mixed with 1 mL lysis buffer containing 40 mM Tris-base, 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2% (v/v) carrier ampholytes pH 3–10 and 65 mM DTT. The homogenates were shaken for 15 min in an ice-water bath and centrifuged at 14000 rpm for 15 min at 4 °C. Protein concentrations of supernatants were determined by the Bradford method.
2-DE, gel staining and image analysis
Before loading for 2-DE, samples were dissolved in 350 μL rehydration buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2% (v/v) IPG buffer, 20 mM DTT, and a trace of bromophenol blue, then centrifuged at 14000 rpm for 5 min. Total protein extracts from control and treated samples were separated through 2-DE. Two protein samples (140 μg) were loaded onto analytical and preparative gels, respectively. Isoelectric focusing (IEF) was carried out on an IPGphor system (Amersham Biosciences) with pH 4–7 IPG strips (18 cm, linear) according to the manufacturer’s instructions. A total of 60 kVh was applied. Then the IPG strips were equilibrated in 3 mL equilibration buffer twice for 15 min. The first equilibration was performed in a buffer containing 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 1% (w/v) DTT, and a trace amount of bromophenol blue. The second equilibration was performed in a buffer modified by 2.5% w/v IAA instead of DTT. The strips were placed on the top of 12.5% SDS-polyacrylamide gels and sealed with 0.5% agarose. Electrophoresis was carried out on a Hoefer SE 600 apparatus (Amersham Biosciences) at 20 °C with the current of 15 mA/gel for 40 min, and then 45 mA/gel for 6 h. The protein spots in gels were visualized by staining with silver nitrate [25]. At least three replicates were performed for each sample. Images of each gel were acquired using Lab-Scan version 3.0 software (GEHealthcare) on an Image-Scanner. Images were analyzed by ImageMaster 2-DE platinum version 5.0 software. The intensity of the protein spots was calculated with PDQuest 8.0 software.
Protein digestion, LTQ-MS/MS and database searching
In order to locate protein spots with different intensity in gels, Coomassie Brilliant Blue G-250 was used to stain the gels for mass spectrometric analysis. The gels were fixed with a buffer containing 40% ethanol and 10% glacial acetic acid for 1 h, washed with double distilled water three times, stained with Coomassie Brilliant Blue G-250 staining solution overnight, and decolorized in a destaining solution for at least 4 h. The significantly altered protein spots were located by comparing gels with control and treated samples side by side. The identified protein spots were cut out from the gel, further destained with 30 mM potassium ferricyanide/100 mM sodium thiosulfate (1:1, v/v) for 20 min, and washed in Milli-Q water until the gels were completely destained. The spots were kept in 0.2 M NH4HCO3 for 20 min and then lyophilized. Each spot was digested in 12.5 ng/mL trypsin with 0.1 M NH4HCO3 overnight. The peptides were extracted with 50% Acetonitrile, and 0.1% TFA three times.
Separation and identification of the digested proteins were conducted on a Finnigan LTQ mass spectrometer (ThermoQuest, San Jose, CA, USA) coupled with a Surveyor HPLC system (ThermoQuest, San Jose, CA, USA). A Microcore RP column (C18 0.15 mm × 120 mm; ThermoHypersil, San Jose, CA, USA) was used to separate the protein digests. Solvent A was 0.1% (v/v) formic acid, and solvent B was 0.1% (v/v) formic acid in 100% (v/v) ACN. The gradient was held at 2% solvent B for 15 min, and increased linearly to 98% solvent B for 90 min. The peptides were eluted from the C18 microcapillary column at a flow rate of 150 μL/min and then electrosprayed directly into an LCQ-Deca mass spectrometer with the application of spray voltage of 3.2 kV and capillary temperature at 200 °C. The full scan was ranged from M/Z 400 to 2000. Protein identification based on MS/MS data was performed with SEQUEST software (University of Washington, licensed to Thermo Finnigan) based on the database of Swiss Port. The species for sequence search is Lepidoptera. Protein identification results were filtered with a stringent filter condition of Xcorr (1 + ≥ 1.9, 2 + ≥ 2.2, 3 + ≥ 3.75) and DelCn (≥ 0.1).
Identification and sequencing of sf-DnaJ1 cDNA
To obtain a full length Sf-DnaJ1 cDNA, total RNA was isolated from Sf9 cells with an E.Z.N.A.™ Total RNA Kit II (OMEGA, USA) according to the manufacturer’s instructions. First strand cDNAs were synthesized using a PrimeScript® 1st Strand cDNA Synthesis Kit (TaKaRa) according to the provided protocol. cDNA of Sf-DnaJ1 was amplified by PCR with the degenerate primers Sf-DnaJ1-F and Sf-DnaJ1-R (Additional file 1: Table S1) and 50 μL reaction mixture contained 0.5 μL template, 1 μL of each 10 mM primer, 0.5 μL Taq DNA polymerase (TIANGEN, Beijing, China), 4 μL of 2.5 mM dNTP mixture (TIANGEN, Beijing, China), and 5 μL 10× Taq Buffer. The PCR program was performed with 32 cycles of 30 s at 94 °C, 30 s at 55 °C and 30 s at 72 °C. The 5’-RACE (SMART RACE, Clontech) and 3’-RACE (TaKaRa) methods were used to fulfill the full-length cDNA of Sf-DnaJ1. To ensure the 5′ and 3′ fragments were cloned from the same gene, specific primers were designed and PCR was used to amplify the coding region of the transcript encoding Sf-DnaJ1.
Quantitative real time PCR
In order to confirm the expression profiles of six identified proteins, quantitative real-time PCR (qRT-PCR) was performed. Total RNA was extracted from Sf9 cells treated with azadirachtin for 24 h and control cells using a Total RNA Kit II (OMEGA, USA). The cDNA for qRT-PCR was synthesized using a PrimeScript™ RT reagent Kit (TaKaRa, Japan), which has a gDNA Eraser to eliminate DNA contamination. qRT-PCR was performed on CFX Connect™ Real-Time System (Bio-Rad, USA) using SsoAdvanced™ SYBR® Green Supermix (Bio-Rad, USA). The PCR was carried out as follows: 95 °C for 3 min for denaturation, 40 cycles of 95 °C for 10 s, 60 °C for 10 s, 72 °C for 30 s, and a dissociation step at the end. GAPDH was used as a reference for normalization. Relative expression levels were calculated by the 2−ΔΔCT method. Primers used in the experiments are listed in Additional file 1: Table S1.
Western blot assays
Cells were collected and washed with PBS. Total cellular proteins were extracted using the CytoBuster™ Protein Extraction Reagent (Novagen, USA) according to the manufacturer’s protocol. Protein concentrations were determined by the Bradford method. Equal amounts of proteins from different samples were separated on a 12% SDS-PAGE gel. Proteins in the gel were then transferred to a polyvinylidene difluoride membrane (PVDF, Millipore, USA). The membrane was washed with TBS for 3 times, incubated with TBS supplemented with 5% fat-free milk at 4 °C overnight, and incubated with HSP 40 antibody, TCTP antibody or GAPDH antibody at room temperature for 2 h. Subsequently, the membrane was washed and incubated with the peroxidase-conjugated secondary antibody at room temperature for more than 2 h. The protein bands were detected by the enhanced chemiluminescence western blot kit (CW0049, CWBIO, Beijing, China) and detected by exposure to X-ray film a dark room.
Double-stranded RNA synthesis and transfection
dsRNA against the Sf-DnaJ1 transcript was synthesized using a T7 RiboMAX™ Express RNAi System (Promega, USA) according to the provided protocol. Template DNA was amplified by PCR with the RNAi primers listed in Additional file 1: Table S1. The dsRNA against egfp, which used as the negative control, was similarly synthesized by the template pEGFP-C and primers in Additional file 1: Table S1. The size and integrity of dsRNAs were checked by agarose gel electrophoresis.
Transfection of Sf9 cells was performed based on the Lipofectin transfection method. Monolayer cultures of Sf9 cells were prepared in 35-mm cell culture dishes (Corning, USA). Transfection was carried out by incubation with 2 mL Hyclone SFX-insect cell culture medium (without FBS) containing 5 μg dsRNAs overnight at 27 °C, followed by incubation in 10 μL lipofectamine 2000 (Invitrogen) for 6 h. The medium was then replaced with a medium containing FBS. After 24 h treatment, the RNAi efficiency was examined based on qRT-PCR and western blot results.
Annexin V-FITC/propidium iodide double-staining and flow cytometry
Anchorage-dependent Sf9 cells treated with 0.75 μg/mL azadirachtin for 24 h were collected by centrifugation at 2000 rpm for 5 min at 4 °C. The cells were then resuspended and washed twice with PBS. The cells were then fixed in 500 μL binding buffer. Prior to cytometry analysis, 5 μL Annexin V-FITC and 5 μL PI were added to the fixed cells which were then incubated in dark for 15 min at room temperature. The cells were analyzed through a flow cytometry with an Ar laser with excitation and emission wavelengths 488 nm and 530 nm, respectively. At least 2.0 × 104 cells were counted in each assay. The Sf9 cells with Sf-DnaJ1 knocked down and GFP control cells were treated with 0.75 μg/mL azadirachtin for 24 h and used for analyses.
Data analysis
Each treatment had three replicates and data were expressed as the mean values ± SEM. One-way ANOVA followed with Duncan’s new multiple range test (DMRT) and student’s t test were conducted during statistical analyses (P < 0.05).