2.1 Reagents and antibodies
Purchased from Sigma (St Louis, MO, USA), 5-aminolevulinic acid (ALA) was dissolved in PBS to a stock concentration of 1M and was stocked at -20°C away from light. Sigma-Aldrich (St Louis, MO, USA) offers paraformaldehyde, bovine serum albumin (BSA), N- acetylcysteine (NAC), diphenyl chloride (DAPI), as well as Anti-GAPDH, and anti-LC3 polyclonal antibody. Goat mouse IgG- IgG-horseradish peroxidase and goat anti-rabbit IgG-horseradish peroxidase were come from Thermo Fisher Scientific.
Antibodies for anti-caspase-3, anti-caspase-9 were purchased from Cell Signaling Technology (Santa Cruz, CA, USA). Human Lamp-2 antibody was obtained from R&D (Minneapolis, MN, USA). PINK1 antibody was from
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Novus Biologicals (Littleton, CO, USA). Anti-Parkin antibody was purchased from Abcam (Cambridge, UK), LC3B polyclonal antibody, Catalase polyclonal antibody, MFN1 polyclonal antibody, SOD2 polyclonal antibody, and FIS1 polyclonal antibody were from Thermo Fisher Scientific (Carlsbad, CA, USA). Immunofluorescent secondary anti-rabbit/mouse IgG was from Jackson Immune Research (West Grove, PA, USA).
2.2 Cell incubation and Sonodynamic therapy
Deriving from a cell library Academy of Sciences (Shanghai), MCF-7 cells were stored in Dulbecco's modified Eagle medium (DMEM, Life Technology, Carlsbad, USA). It was added with 10% fetal bovine serum (FBS, Life Technology), 100 units / ml penicillin and 100 ug / ml streptomycin. At a temperature of 37 ℃, MCF- cells were stored in 95% air and 5% CO2
humidified chamber. MCF-7 cells were digested with 0.25% trypsin and passaged at 80% confluence. Randomly, MCF-7 cells were classified into four groups: (1) control, (2) ultrasound alone (ultrasound), (3) ALA alone, and (4) ALA plus ultrasound (SDT). 1mM ALA was used to incubate cells in ALA and ALA-SDT groups. And 4h was taken to load drug in each group with DMEM medium containing 10% FBS. The cells of the ultrasound group and
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the SDT group were placed under an ultrasonic wave with an intensity of 0.25 W/cm2 at a frequency of 1.0 MHz burst mode with the pulse repetition frequency of 100Hz and duty cycle of 10% for 10 min. The ultrasonic generator and a power amplifier used in this study were purchased from Tektronix (Oregon AFG 3251) and AR Inc (Model 500A250C).The planar transducer has a diameter of 35 mm. For irradiation, an ultrasonic couplant was used to fill the spacing between the transducer and the cell culture plate.
By this way, transmission of ultrasound can be promoted. After the treatment, cells used for different analyses were incubated in fresh medium for different times (2 hours, 4 hours, 12 hours).
2.3 Determination of cell viability
Cell viability was determined at various time points after ALA-SDT treatment using Cell Counting Kit-8 (Sigma-Aldrich) with reference to the manufacturer's guidelines. Our method is to incubate the cells with 96-well plates at a density of 5000 cells per well. The cells are incubated in 100-μl medium for 24 hours. And then CCK-8 reagent 10μ1 per well at 37 ° C and 5% CO2 were added. Cytotoxicity was measured in 1 hour. At 450 nm, the absorbance of the sample relative to the blank control was determined as the
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detection wavelength. Compared with the control group, changes in cell viability were observed.
2.4 Apoptosis assay was detected by flow cytometry.
Being bred at a density of 5× 105 cells in 6-cm dishes, cell culture time was 24h. Referring to the manufacturer's guidelines, cell apoptosis at various time point after ALA-SDT was measured by Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific). After being collected, cells were incubated with 1 µL of the PI working solution and 5 µL of the annexin V conjugate for 15 minutes at room temperature. And then it will be analyzed through the BD Accuri C6 Software (Becton-Dickinson, USA) and FACS Calibur flow cytometer.
2.5 Detection of mitochondrial membrane potential
JC-1(Sigma-Aldrich) was utilized to determine mitochondrial membrane potential. Briefly, before being washed with FACS buffer, at 37 ° C cells were dark staining with 2.5μM JC-1 for 30 min. FACS Calibur flow cytometer and BD Accuri C6 Software (Becton-Dickinson, USA) are responsible for analyzing the data. Excitation and emission settings JC-1 monomers were 488 nm and 515 ~ 545 nm (FL1 channel); excitation and
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emission settings JC-1 aggregations were 488nm and 564 ~ 606nm (FL2 channel).
2.6 ATP generation test
Consistent with the manufacturer's instructions, firefly luciferase-based ATP assay kit was utilized for measuring cellular ATP production (Thermo Fisher Scientific). After lysis centrifugation for 5 min at 12000 g, the 10μ1 cell lysate and 100μl ATP detection fluid were mixed in 96 well white plate.
These data are coordinated by a microplate reader Biotek (Biotek Tools Inc., Winooski, Vt, USA) collection. Protein concentration in the cell lysate was measured by BCA kit. Data was evaluated using by ratio of cellular ATP levels and protein concentration.
2.7 Mitochondrial dynamic assay
Consistent with the manufacturer's instructions, mitochondria were tracked by CellLight Mitochondria-GFP (Thermo Fisher). In a word, the reagent was involved in the incubation with cells all the night, mitochondria-GFP behavior can be traced in live cells. After being treated by ALA-SDT,
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cells were observed using laser-scanning confocal microscopy with X63 objective (Nikon, Tokyo, Japan).
2.8 Immunofluorescence microscopy of mitophagy
According to the standard guidelines, mitochondrial autophagy and lysosomes colocalization are the determining factors of mitophagy (Ding and Yin 2012). Each 35mm confocal culture dish (SPL Life Sciences, Korea) was inoculated with 1 x 105 cells for 24 hours. 100 nm Mito Tracker Deep Red stains cells (Thermo Fisher) for 15 min at a temperature of 37°C. Then, PBS was used to wash cells and 4% PFA was utilized to fix them for 15min at room temperature. After being fixed, another night was taken for further incubation.
Antibodies against autophagosome marker LC-3 or LAMP 2 and Alexa Fluor 488- or 594-conjugated secondary antibody will be added. 5% bovine serum albumin (Sigma) was used to dilute the antibodies. The nuclei were dyed for 10min at room temperature. Laser-scanning confocal microscopy with X63 objective (Nikon, Tokyo, Japan) was used to visualize samples. Under each condition, 20 to 30 cells were captured. The number of mitochondria colocalized with LC3 or lamp 2 was quantified.
2.9 Cell lysis and Western blot analysis
In order to get cell lysates, cold PBS was applied to wash the cells. Then,
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the cells were lysed in RIPA in lysis buffer for 10 min on ice. (50mM Tris-HCl, pH 7.4, 150mM NaCl，1% NP-40，0.5% sodium deoxycholate，0.1%
SDS). The buffer also contains protease inhibitor mixture (Roche, Basel, Switzerland) and a phosphatase inhibitor mixture (Roche). 10%
polyacrylamide gels were loaded by the complexes which had went through the quantification and denaturation. Then the complexes moved onto polyvinylidene difluoride (PVDF) membranes. Membranes were then blocked with 5% milk diluted in PBS containing 0.05% Tween 20 (PBST) for 1 h and then immunoblotted at 4°C overnight with the specified primary antibodies.After washing in PBST for 10 min three times, the membranes was incubated 1h at room temperature with the corresponding secondary anti-rabbit/mouse IgG. After treatment of these members with ECL reagent (USA, Bio-Rad), a FluoChem E Imager (Protein-simple, USA) was used to observed the samples. Image J was applied to quantify the density of protein. The GAPDH protein is considered an internal standard for semi-quantitative.
2.10 RNA interference
Thermo Fisher offers the small interfering RNA (siRNA) duplexes. In line with the manufacturer’s instructions, LipofectamineTM 2000 was used to conduct iRNA. To summarize, 1 x 105 cells were inoculated and incubated for
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24 hours in a 35mm confocal culture dish (SPL Life Sciences, Korea). 20 μM synthesized siRNA transfected cells to target at Parkin. The siRNA and LipofectamineTM 2000 were respectively diluted in serum-free DMEM and incubated for 5 min at room temperature. The two diluted solutions were then softly mixed, incubated for 20 minutes, and finally added to the cells.
Inhibition was observed after 24h after transfection, the expression of Parkin, analyzing the Parkin expression with Western blot.
2.11 Determination of cell and mitochondrial ROS amount
DCFH-DA(Sigma-Aldrich) was used to measure Intracellular ROS. In brief, MCF-7 cells were incubated with 10 mM DCFH-DA diluted with DMEM at 37°C for 20 min, and then washed with PBS for three times. The labeled cells were trypsinized and analyzed by flow cytometry. Referring to the manufacturer's instructions, the production of mitochondria-derived reactive oxygen species (mROS) is measured by MitoSOX (Invitrogen, USA).
10μM of MitoSOX were applied for the incubation of cells at 37°C for 20 min.
Laser-scanning confocal microscopy with X63 objective (Nikon, Tokyo, Japan) was used to observe labeled cells which were washed with PBS three times. Finally, Nikon NIS-Elements software serve as the tool for analysis.
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2.12 Statistical analysis
GraphPad Prism software was applied to do statistical analysis. Image processing is handled by Image J, Photoshop CS, and Illustrator CS software in accordance with general guidelines. All data are expressed as mean ± SEM, using a two-tailed Student’s t-test or one-way ANOVA. It is considered that P-values <0.05 were of significance statistically.