Primary liver cells were isolated from BALB/c mice by collagenase B according to the manufacturer's instructions (Worthington Biochemical, Lakewood, NJ) and cultured as described (Zurlo and Arterburn, 1996
). Primary cultures of heart cells were dissociated from ventricles of 1- or 2-d-old neonatal Sprague Dawley rats (Harlan, Indianapolis, IN) by enzymatic digestion of 0.1% trypsin overnight (US Biochemicals, Cleveland, OH) and then 0.1% collagenase (Worthington Biochemical) for dissociation as previously described (Iravanian et al., 2003
). Peritoneal macrophages were isolated from BALB/c mice as previously described (Hu et al., 2009
). NIH 3T3 cells, human cervical cancer HeLa cells, and Mpf brain cells (CRL 1656) were purchased from the American Type Culture Collection (Manassas, VA). The cells were cultured in DMEM/F-12 (DMEM:nutrient mixture F-12) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Carlsbad, CA) at 37°C under an atmosphere of 5% CO2
/95% air. Cells were seeded onto tissue culture plates for 2 d with 70% confluence before being subjected to each experiment. The medium was changed every 24–36 h.
Unless specifically mentioned, apoptosis in the primary mouse liver cells and primary rat heart cells was induced by 5-h exposure to 4.5% ethanol (Scharlau, Barcelona, Spain), HeLa cells by 5-h exposure to 4.3% ethanol, and NIH 3T3 cells by 20-h exposure in 10% DMSO (Sigma-Aldrich, St. Louis, MO) in culture medium (vol/vol). Mpf cells were induced by 50-h incubation in 2 μM jasplakinolide (Invitrogen, Carlsbad, CA) and primary mouse macrophages by 24-h incubation in 1 μM cucurbitacin I (ChromaDex, Irvine, CA) in culture medium. These conditions were chosen because they represented the lowest concentration of inducer that caused >90% of cells to undergo apoptosis. For removal of apoptotic inducers, cells were washed three times with culture medium and then cultured for the period of time indicated in the individual experiments.
Immunocytochemistry and fluorescence and confocal microscopy
Mitochondria and nuclei were stained in living cells with 50 nM Mitotracker Red CMXRos and 250 ng/ml Hoechst 33342 (Invitrogen), respectively, for 20 min in culture medium. Cells with active endocytosis were labeled by green fluorescence–emitting Quantum Dots from Qtracker 525 Cell Labeling Kit (Invitrogen) as described (Jaiswal et al., 2003
). ApoAlert pCaspase3-Sensor Vector (BD Clontech, San Jose, CA) was transfected to HeLa cells using X-tremeGENE 9 DNA Transfection Reagent (Roche, Indianapolis, IN). Cell surface membrane phosphatidylserine (PS) was detected by using Annexin V Apoptosis Detection Kit according to manufacturer's instructions (BioVision, Milpitas, CA). The cells were fixed with 3.7% (wt/vol) paraformaldehyde in phosphate-buffer saline (PBS) solution for 20 min at room temperature, and incubated with 0.1% Triton X-100 (vol/vol) (Sigma-Aldrich) for 10 min before immunostaining. Endogenous AIF and EndoG were stained with anti-AIF and anti-EndoG primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and conjugated with green fluorescent Alexa Fluor 488 and red fluorescent Alexa 594 anti–rabbit immunoglobulin G secondary antibodies with Zenon Tricolor Labeling kit (Invitrogen), respectively. Cell images were captured with a monochromatic CoolSNAP FX camera (Roper Scientific, Tucson, AZ) on an inverted fluorescence microscope Cell Observer Z1 using a 63×, numerical aperture (NA) 1.4 plan-Apochromat objective or LSM 710 on an upright microscope Axio Examiner using a 40×, NA 1.1W Corr LD C-Apochromat objective (Carl Zeiss, Jena, Germany). Images were analyzed by using AxioVision 4.2 software (Carl Zeiss).
Real-time live-cell microscopy
Cells were cultured in CO2-independent medium (Invitrogen) on a glass-bottom culture dish (MatTek Corporation, Ashland, MA) or a thermo-cell culture FCS2 chamber (Bioptechs, Butler, PA), which was mounted onto the adapter in the stage of an inverted fluorescence microscope. Ethanol (4.5% for liver, 4.3% for HeLa cells [vol/vol]) in culture medium was introduced to the cells through perfusion tubes (Bioptechs), which were connected to the cell chamber. The ethanol was removed, and fresh medium was then introduced to the chamber through these tubes after apoptotic induction. Fluorescence signals of mitochondria and nuclei were visualized by fluorescence with excitation at 561 and 405 nm, respectively, and cell morphology by differential interference contrast (DIC) or phase contrast microscopy. Time-lapse images were captured by Cell Observer Z1 (Carl Zeiss) with a monochromatic CoolSNAP FX camera (Roper) or Evolve 128 EMCCD (Photometrics, Tucson, AZ) using a 63×, NA 1.4 Plan-Apochromat objective or a 40×, NA 0.95 Corr Plan-Apochromat objective (Carl Zeiss), a BioStation IMQ time-lapse imaging system (CELL-S2) using a 40×, NA 0.8 Plan-Apochromat objective (Nikon, Melville, NY), or a C2 Confocal on a TiE Invert microscope using a 60×, NA 1.4 Plan-Apochromat objective (Nikon). Images and heat map were analyzed by using AxioVision 4.2 software.
Western blot analysis
Approximately 3 μg of protein from total cell lysate per lane was separated on a 12% SDS–PAGE gel and transferred onto a Hybond ECL membrane (Amersham Biosciences, Chalfont St Giles, United Kingdom). After blocking, the membrane was incubated overnight at 4°C with primary antibody detecting targeted protein as stated in the text with 1:1000 dilution, followed by another hour of incubation with the corresponding horseradish peroxidase–conjugated secondary antibody (Bio-Rad, Hercules, CA) at room temperature with 1:5000 dilution. Primary antibodies used were as follows: anti-caspase-3, anti-PARP (Cell Signaling Technology, Danvers, MA), and anti-ICAD (BD PharMingen, BD Biosciences, Le Pont de Clax, France). The signal from the secondary antibody was detected with the ECL Western blotting detection system (Amersham-Pharmacia Biotech, GE Healthcare Bio-Sciences, Piscataway, NJ).
Single-cell gel electrophoresis (comet) assay
Comet assay was performed by using the Trevigen Comet Assay kit (Trevigen, Gaithersburg, MD) according to manufacturer's instructions. Alkaline electrophoresis of gelled slides was performed using Ready Sub-Cell GT Cells (Bio-Rad) on ice with 20 V and 200 mA for 30 min. The current was adjusted by the volume of the buffer in the gel tank. The DNA was visualized by SYBR Green staining (Trevigen), followed by fluorescence microscopy.
Cytokinesis-block micronucleus assay
Cells were grown on glass coverslips (Marienfeld, Lauda-Künigahofen, Germany) with 70% confluence in six-well cell culture plates (Nunc, Roskilde, Denmark) and were induced to apoptosis as described in the section Apoptotic inductions
. To study genomic damage in apoptotic cells that reversed apoptosis and proliferated, the apoptosis-induced cells were washed and cultured for 16 h in fresh medium that contained cytokinesis-blocking cytochalasin B (3 μg/ml; Sigma-Aldrich). Cells without apoptotic induction served as control. The cells were then fixed by incubating in methanol/acetic acid (5:1, vol/vol) twice for 15 min, followed by overnight fixation at 4°C. After washing three times with PBS, the fixed cells were stained for nuclei by incubation with 250 ng/ml Hoechst 33342 in PBS for 20 min at room temperature. Slides were then prepared as described for fluorescence microscopy and observed under a 63× objective to image micronucleus. The micronuclei in the cytokinesis-blocked cells were scored as described (Fenech, 2007
). Only binucleated cells were scored, so as to include only cells that divided once after addition of cytochalasin B. The two main nuclei should have a clear boundary from each other, and micronuclei were counted only when clear boundary from the main nuclei was observed. The diameter of a micronucleus should lie between 1/16 and 1/3 that of the main nuclear diameter. Triplicates were performed, with >100 cells for each condition per set.
Metaphase chromosome spreads were prepared as described (MacLeod et al., 2007
) with modifications. Briefly, cells were arrested at metaphase by adding colchicine (Sigma-Aldrich) at a final concentration of 1 μg/ml into growing culture for 6 h. The arrested cells were then collected by trypsinization with immediate neutralization with cell culture medium and then followed by 5 min of centrifugation at 400 × g
. After discarding the supernatant, we loosened the cell pellet by gentle flicking in residual medium. To swell the mitotic cells, we incubated the cell suspension in hypotonic buffer, potassium chloride (5.59 g/l in double-distilled water), and sodium citrate (9.0 g/l in double-distilled water) in 1:1 (vol/vol), for 8 and 15 min for NIH 3T3 and primary liver cells, respectively, at 37ºC. The cells were then pelleted at 400 × g
for 5 min to remove the hypotonic buffer. The cells were then fixed by gently adding freshly prepared ice-cold fixative (methanol/acetic acid, 3:1, vol/vol) to the pellet while agitating the centrifuge tube for the whole time so as to prevent cell clumps formation and ensure thorough mixing. The fixative was changed once, and then the cells were fixed overnight at 4ºC. Next, the cells were concentrated in fixative of a volume such that the suspension became slightly cloudy for optimal cell concentration. To spread the metaphase of the fixed cells onto slides, we dropped the cell suspension from height onto a chilly, precleaned SuperFrost Plus microscopic slide (Gerhard Menzel, Braunschweig, Germany) slightly sloped on a freezer block. Then the slides were breathed on to enhance spreading and were mounted with 4′,6-diamidino-2-phenylindole (DAPI)/Antifade kit (MetaSystems, Altlussheim, Germany) after drying. The metaphase chromosomes of metaphase-arrested cells were identified and captured by an automated cytogenetic scanner workstation (MetaSystems) for analysis. Only metaphases of distinctly separated chromosomes and of chromosome spreading patterns from one nucleus were counted in order to avoid overlapped metaphases. Three replicates of >100 metaphases each were counted for the presence of radial configurations in each corresponding metaphase spread for chromosomal abnormality.
For the focus formation assay, cells were seeded in 10-cm2
culture dishes (Nunc) to reach 70% confluence. They were induced and washed as described. Then the culture medium was changed every 3 d. After 3 wk of culture, morphologically transformed foci whose diameter exceeded 0.5 mm were counted. The assay was performed three times. From each replicate, at least five transformed foci were isolated by picking with sterile pipette tip and were then cultured for soft agar assay. Anchorage-independent colony formation of NIH 3T3 cells undergoing anastasis was determined as described previously (Cifone and Fidler, 1980
) with some modifications. Briefly, the cells were harvested by trypsinization. A total of 3 × 103
cells were resuspended in 1.5 ml of complete cell culture medium containing 0.3% agarose. The suspensions were cultured in single wells of six-well cell culture plates (Nunc) above a layer of solidified 0.5% agarose in the medium. After incubation at 37°C under an atmosphere of 5% CO2
/95% air for 5 wk, plates were stained with 0.5 ml of 0.005% crystal violet solution (Sigma-Aldrich) for 1 h before being subjected to microscopy.
New RNA detection
For newly synthesized RNA detection, cells were incubated in the presence of 50 μM 4-thiouridine (s4U; Sigma-Aldrich) for 1 h and total RNA was extracted by TRIzol (Invitrogen). The RNAs were then subjected to biotinylation as described (Zeiner et al., 2008
). The biotin-labeled RNAs were agarose electrophoresed, transferred to nylon membrane (Bio-Rad) with Trans-Blot SD DNA/RNA Blotting Kit (Bio-Rad), and then detected by chemiluminescence using LightShift Chemiluminescent EMSA Kit (Pierce, Rockford, IL).
Microarray and gene expression data analysis
Mouse primary liver cells were treated with 4.5% ethanol for 5 h (R0) and then washed and cultured in fresh medium for 3 (R3), 6 (R6), 24 (R24), and 48 (R48) h. The untreated cells were used as control (Ctrl). Total RNA was isolated and purified by RNeasy Mini Kit (Qiagen, Cologne, Germany). As a first level of quality control, to detect possible batch effects or sample outliers, we performed principal component analysis with Partek Genomics Suit 6.5 (Partek, St. Louis, MO) on log2-normalized signal values for all samples; biological replicate samples were observed to cluster together. Furthermore, an analysis of variation was run on all data, including variables for cell sample, batch, and error, in which analysis also showed an excellent ratio of signal (biological variation) to noise (batch and error). The RNA was subjected to reverse transcription using SABiosciences C-03 RT2 First Strand Kit (SABiosciences-Qiagen, Frederick, MD). These cDNA samples were analyzed on the Illumina MouseWG-6 v2.0 Expression BeadChip (Illumina, San Diego, CA).
Processed Illumina signal value data were imported into the Partek and Spotfire DecisionSite 9.1 (TIBCO, Palo Alto, CA) platforms for evaluation for expression fold change at the gene level between time points and for fold change across time when compared with a common starting point. First, all signal values were converted into log2 space and quality control tests run to ensure data integrity. Signals for the three biological replicates at each time point were taken together for comparison to other time points; Student's t
test used to determine statistical significance as p values, and fold change was based on averaged values. For our time-course analysis, all time points were compared with time point R0, the time at which ethanol treatment ended. Functional Gene Ontology (The Gene Ontology Consortium, www.geneontology.org
) and pathway analyses were run with Spotfire's Gene Ontology Browser and Ingenuity Pathway Analysis (Ingenuity Systems, www.ingenuity.com
) software, respectively.
Total RNA was isolated and purified by RNeasy Mini Kit (Qiagen, Cologne, Germany), and 1.5 μg of the total RNA was reverse transcribed into cDNA via the M-MLV reverse transcriptase (Invitrogen) with oligo-dT as primer. RT-PCR was performed on an IQ5 machine (Bio-Rad) using SYBR GreenER qPCR SuperMix (Invitrogen) in 25 μl of reaction with the following PCR cycle parameters: 10 min at 95°C (pre-denaturation and hot start); 40 cycles of 35 s at 95°C; 35 s at 58°C; 30 s at 72°C (denaturation/annealing/amplification). The following primers were used for detection of their corresponding mRNA. Bcl2 forward primer sequence, 5′-CCT GTG GAT GAC TGA GTA CC-3′; reverse primer sequence, 5′-GAG ACA GCC AGG AGA AAT CA-3′ (Sigma-Aldrich). Xiap forward primer sequence, 5′-CTG AAA AAA CAC CAC CGC TAA C-3′; reverse primer sequence, 5′-CTA AAT CCC ATT CGT ATA GCT TCT TG-3′. Mdm2 forward primer sequence, 5′-CGG CCT AAA AAT GGC TGC AT-3′; reverse primer sequence, 5′-TTT GCA CAC GTG AAA CAT GAC A-3′. Hsp90aa forward primer sequence, 5′CTC CAA TTC ATC GGA CGC TCT G 3′; reverse primer sequence, 5′ TCA AGT CGG CCT TGG TCA TTC C 3′. Gapdh forward primer sequence, 5′-TGC CTC CTG CAC CAC CAA CT-3′; reverse primer sequence, 5′-CGC CTG CTT CAC CAC CTT C-3′. All RT-PCR assays were completed in triplicate, and the threshold cycle of each reaction was converted to DNA equivalent by reading against its corresponding standard curve generated by amplifying dilutions of cDNA containing the relevant target sequences. The relative mRNA expression levels of the target genes were normalized to the mean of Gapdh, which served as the internal control.
Immediately after removal of apoptotic inducers, specific inhibitor targeting BCL-2 (ABT 263, 1 μM; Selleck Chemicals, Houston, TX), XIAP (Embelin, 20 μM; Sigma-Aldrich), MDM2 (MDM2 inhibitor, 20 μM; Sigma-Aldrich), or HSP90 (17-allylaminogeldanamycin, 0.5 μM; Sigma-Aldrich) was applied to the cells together with fresh culture medium. A mock experiment was also performed in the untreated cells.
Statistical comparison was performed using two-tailed (unless stated otherwise) Student's t test (except in microarray and gene expression data analysis, as described in the corresponding section). Differences were considered to be significant when the p value was <0.05.