2.1 Chemicals and reagents
The Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin (P/S) solution were purchased from Gibco (Gibco, New York, NY, USA). Glucose was purchased from Sigma-Aldrich (St. Louis, MO, USA). Autophagy inhibitors including 3-Methyladenine (3-MA, S2767) and Z-DEVD (S7312) were bought from Selleck (Shanghai, China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Dojindo, Kumamoto, Japan). DMSO, MDC staining kit, trypan blue and protease inhibitor were purchased from Sigma (Darmstadt, Germany).
2.2 Cell culture
The embryonic rat heart–derived myogenic cell line H9c2 were purchased from the American Type Culture Collection (ATCC), and the cells were maintained in DMEM containing 10% FBS (V/V), 100 U/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 5% CO2 at 37°C, as described previously [12, 13]. All cells used in the present study were below the 35th passage.
2.3 Plasmids, Ad vectors, and transfection
Adenovirus (Ad) vectors including Ad-Sphk1 and Ad-shSphk1 were constructed by using an improved in vitro ligation method [14]. Briefly, FLAG epitope–tagged WT human sphingosine kinase 1 (Sphk1) cDNA (GenBank accession no. AF200328) was sub-cloned into pcDNA3* vector, followed by generating adenoviruses. All Ad vectors (Ad-Sphk1 and Ad-shSphk1) were purified as described previously [14]. The virus particles (VPs) were measured using an Adeno-X-rapid titer kit (Clontech, Mountain View, CA, USA).
Before transduction, H9c2 cells cultured in T75 flask were passaged into six-well plates (2x105 cells/well), and cells were cultured overnight to reach 70–80% confluence. Afterwards, adenoviruses of Ad-Sphk1 and Ad-shSphk1 with multiplicity of infection (MOI) of 100 were used to transduce cells, followed by being selected in growth medium containing 1 mg/mL G418. All experiments were conducted in the absence of serum at 50% confluence. The transduction was verified by western blot and observation under fluorescence microscope.
2.4. The mRNA expression using quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from H9C2 cells using TRIzol (Invitrogen) according to the manufacturer's instructions [15, 16]. The RNA reverse transcript (RT) was executed using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech, Beijing, China) following the manufacturer’s protocol and 500 ng RNA was used to generate cDNA. The cDNA products were subjected to qRT-PCR using TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China) according to the manufacturer’s protocol. The protocol for thermal cycling consisted of 60 s at 95 °C, followed by 40 cycles of 15 s at 95 °C and 60 s at 63 °C. Relative mRNA levels of target genes were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results are based on cycle threshold (Ct) values. We calculated differences between the Ct values for experimental and GAPDH and graphed as a percent of each RNA to the calibrator sample. All primers used in the study were listed in Table 1.
2.5. Western blot
The total protein was extracted with RIPA solution from in vitro cultured cells, and protein samples were quantified using BCA Protein Assay Kit (Beyotime Biotechnology). The protein (20 μg) was subjected to 10% SDS-PAGE gel electrophoresis, followed by transferring to a PVDF membrane. Then, the PVDF membranes containing protein were blocked using blocking buffer (Beyotime Biotechnology), followed by being washed for 3 times with PBS containing 0.5% TWEEN-20 (PBST). Afterwards, PVDF membranes were incubated with primary antibodies, including rabbit anti-Sphk1 (1: 1000, 10670-1-AP, Proteintech), anti-Beclin1 (1:1000, bs-1353R, Bioss, Beijing, China), anti-p-LC3B (1:1000, BM4827, Bioss, Beijing, China), and anti-GAPDH (1: 1000, ab9845, Abcam) overnight at 4°C. The next day, membranes were washed for 3 times with PBST, 5 min each time, followed by incubation with anti-rabbit IgG-HRP secondary antibody (1: 5000, Proteintech, Wuhan, China) at room temperature for 1 h. Then, the blocks were washed with PBST for 5 min, followed by imaging with ECL method (Beyotime Biotechnology).
2.6. Lentiviral expression of green fluorescence protein bound to LC3
H9c2 Cells were cultured on a round cover slide on the bottom of well of a 6-well-plate with culture medium and transduced with lentiviral particles carrying a construct of TagGFP2-LC3 driven by the elongation factor-1 promoter (Millipore LentiBrite) for 48 h, followed by replacing cell culture medium containing drugs and treated for indicated time period. Green punctate representing autophagy was assessed by taking images using fluorescence microscope (Mshot, Guangzhou, China).
2.7. Monodansylcadaverine (MDC) staining
To assess autophagy, H9c2 Cells (105 cells/well) were plated in wells of 6-well plates and were treated with indicated treatments for indicated time. Then, cells were subjected to MDC staining buffer for 45 min at room temperature (Nanjing KeyGen Biotech co., Ltd.) according to the manufacturer's protocol. Subsequently, the fluorescence was measured using a fluorescence microscope (Mshot, Guangzhou, China). The activated autophagosome represented acidic vesicles, which was tested by measuring the level of green fluorescence.
To further measure autophagy, after that cells were stained using MDC, the cells were analyzed by a flow cytometry (Altra; Beckman), and 10000 events were recorded per sample at wave length of 355 nm, and data were analyzed by Flow Jo software (Version 7.6.1).
2.8. Cell vitality measurement
Cell growth and vitality were evaluated by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5 -di-phenytetrazoliumromide (MTT) assay or trypan blue according to the manufacturer's protocol (Nanjing KeyGen Biotech co. Ltd.). Briefly, Cells (103/well) were seeded into wells of 96-well plates and cultured for 24 h. Then, cells were subjected to the indicated treatments for indicated time. Afterwards, cell vitality was exanimated at 490 nm on a microplate reader (Synergy™ 2; BioTek Instruments, Inc.) or cell counter (Thermo Fisher).
2.9. Statistical analysis
All data were analyzed using GraphPad software (version: GraphPad Prism5). Data are presented as mean ± SEM and the standard errors of the mean in this study were carried out in triplicates. Statistical comparisons between 2 groups were performed by t test. P < 0.05 was considered to be a statistically significant difference.