Reagents
StemFit AK03N was obtained from Ajinomoto Healthy Supply Co., Inc. (Tokyo, Japan). iMatrix-511 was obtained from Matrixome Inc. (Osaka, Japan). A 10-mmol/L Y-27632 Solution, D-PBS (-), and 0.5 mol/l-EDTA Solution (pH 8.0) were obtained from Nacalai Tesque (Kyoto, Japan). TrypL™ Select Enzyme (1X) and CTS TM Essential 8 TM Medium and TrypL™ Select Enzyme (1X) were obtained from Thermo Fisher Scientific K.K. (Kanagawa, Japan). Recombinant human MFG-E8 was purchased from R&D Systems (Minneapolis, MN, USA). GLS250 Gelatin Solution (1.0 mg/g) was purchased from Nitta Gelatin Inc. (Osaka, Japan). Kyoto probe 1 (KP-1) was obtained from Goryo Chemical, Inc. (Sapporo, Japan). Human GloLIVE TRA-1-60 (R) NorthernLights™ NL557-conjugated Antibody was obtained from R&D Systems, Inc. Cellstain®-Hoechst 33342 solution was obtained from Dojindo Laboratories (Kumamoto, Japan).
Preparation of cell culture plates
Regarding the coating of iMatrix-511 on each well/6-well plate, the following reagents were added per well: 9.6 µl of iMatrix-511 (0.5 µg/µL) + 1.5 mL PBS. The medium supplemented with gelatin and MFG-E8 was prepared by mixing 0%-10% of GLS250 Gelatin Solution (1.0 mg/mL) and 0–2 µg/mL of recombinant human MFG-E8 (50 µg/mL) into the medium.
Maintenance culture of hiPSCs
hiPSC line 201B7 and 15M66 were obtained from the CiRA Foundation (Kyoto, Japan). To culture iPSCs, a publicly available method (CiRA_Ff-iPSC_protocol_Eng_v140310) was used (https://www.cira.kyoto-u.ac.jp/j/research/img/protocol/Ff-iPSC-culture_protocol_E_v140311.pdf).
NGS
The analysis was carried out as a multi-omics analysis work provided by Promega K.K. (Tokyo, Japan). Samples of 1 × 106 cell pellets were prepared according to the TRIZOL Reagent (Thermo Fisher Scientific K.K., Kanagawa, Japan) protocol. In brief, samples were centrifuged at 12,000 g for 10 min at 4°C. The supernatant was then collected, and the volume was measured. An equal volume of ethanol was added to the sample and stirred by mixing with a vortex mixer. The sample was then applied to a Zymo-Spin IC Column and centrifuged at 12,000 g for 30 seconds at room temperature. RNA wash buffer (400 µL) was applied to the column and further centrifuged at 12,000 g for 30 seconds at room temperature. The flow-through was discarded, and a mixture of 35 µL Digestion buffer and 5 µL DNase I (6 U/µL) was added to the column. The column was allowed to stand at room temperature for 15 minutes. RNA pre-wash buffer (400 µL) was then applied and centrifuged at 12,000 g for 30 seconds at room temperature. The flow-through was again discarded, and 400 µL of RNA pre-washed buffer was applied once more to the column and centrifuged at 12,000 g for 30 seconds at room temperature. The flow-through was again discarded, and 700 µL of RNA wash buffer was added to the column and centrifuged at 12,000 g for 2 minutes at room temperature. The column was then placed in a new tube, and 15 µL of DNase/RNase-Free Water was applied to the column and centrifuged at 12,000 g for 30 seconds at room temperature to collect the flow-through. A total of 1.5 µL of the resulting RNA solution was used to measure the concentration by absorbance. The concentration of each RNA was also determined by a fluorescence method.
The RNA Integrity Number (RIN; calculated from the electrophoresis image of the sample total RNA) of the obtained RNA was measured using an Agilent 2100 Bioanalyzer (G2939A; Agilent Technologies, Santa Clara, CA, USA), with a maximum score of 10 points, and the closer the score is to 0, the more degraded the RNA. As the reagent kit, an Agilent RNA6000 Nano kit (5067 − 1511; Agilent Technologies) was used, and as the analysis software program, the 2100 expert software program, version B.02.07 (Agilent Technologies) was used.
miRNA-Seq data analyses
The analysis was carried out as a multi-omics analysis work provided by Promega K.K. (Tokyo, Japan). Library preparation was performed using a reagent kit, and the NEBNext Small RNA Library Prep Set for Illumina was used for NGS (E7330S; New England BioLabs, Inc., Ipswich, MA, USA). In brief, using 1,000 ng of RNA, we added an adapter to the 3' and 5' ends of the RNA sample. We then synthesized cDNA, conducted 12-cycle PCR, extracted the miRNA fraction using Blue Pippin (Sage Science, Inc., Beverly, MA, USA), and conducted automated DNA fragment gel extraction.
The following equipment and reagents were used for sequencing: NextSeq500 (Illumina, Inc., San Diego, CA, USA) and NextSeq 500/550 High Output Kit v2.5 (75 Cycles) < 20024906> (Illumina, Inc.). NGS was performed using a NextSeq500 as follows: (1) addition of sequencing reagents, (2) single-base elongation reaction, (3) removal of unreacted bases, (4) incorporation of fluorescent signal, (5) removal of protecting group and performance of fluorescence imaging. These steps were repeated for 75 cycles.
The primary data analysis method was to create fastq files from the obtained reads using the bcl2fastq software program (Illumina, Inc.).
3' RNA-Seq data analyses
The analysis was carried out as a multi-omics analysis work provided by Promega K.K. (Tokyo, Japan). Library preparation was performed using the QuantSeq 3’mRNA-Seq Library Prep Kit for Illumina (FWD) < 015.384> (Lexogen GmbH). The QuantSeq 3'mRNA-Seq Library Prep Kit was used to prepare libraries for NextSeq NGS. In brief, using 500 ng of total RNA, we synthesized cDNA using oligo(dT) primers, removed template mRNA, conducted double-stranded DNA synthesis using random primers, conducted 12-cycle PCR using adapters for the Illumina sequencer, and conducted purification by beads.
The following equipment and reagents were used for sequencing: NextSeq500 (Illumina, Inc.) and NextSeq 500/550 High Output Kit v2.5 (75 Cycles) < 20024906> (Illumina, Inc.). NGS was performed using a NextSeq500 as follows: (1) addition of sequencing reagents, (2) single-base elongation reaction, (3) removal of unreacted bases, (4) incorporation of fluorescent signal, (5) removal of protecting group and performance of fluorescence imaging. These steps were repeated for 75 cycles.
The primary data analysis method was to create fastq files from the obtained reads using the bcl2fastq software program (Illumina, Inc.).
Standard DIA Proteome Analysis
The analysis was carried out as a multi-omics analysis work provided by Promega K.K. (Tokyo, Japan). Samples of 1 × 106 cell pellets were prepared according to the TRIZOL Reagent (Thermo Fisher Scientific K.K.) protocol. In brief, chloroform was added to the sample containing Trizol, mixed, and centrifuged (15,000 g, 4°C, 15 min). The aqueous layer was removed, acetonitrile was added to the remaining Trizol solution, and the protein was precipitated. We then added 100 mM Tris-HCL pH 8.5, 2% SDS to the precipitate and dissolve the protein using a sample-sealed ultrasonic disruption machine. The protein concentration was measured by a bicinchoninic acid (BCA) assay and adjusted with 100 mM Tris-HCL pH 8.5, 2% SDS to bring the protein concentration to 0.5 µg/µL. To cleave the S-S bond of the protein, tris(2-carboxyethyl) phosphine (TCEP) was added to the protein lysate (20 µg protein) to a final concentration of 20 mM and incubated at 80°C for 10 min. To alkylate cysteine residues, iodoacetamide (IAA) was added to a final concentration of 30 mM and incubated at room temperature (light-shielded) for 30 minutes. Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (Hydrophylic) and Sera-Mag Carboxylate-Modified Magnetic Particles (Hydrophobic) (Cytiva, Tokyo, Japan) were mixed 1:1 (v/v) and washed 3 times with distilled water to a concentration of 15 µg solids/µL in distilled water (SP3 beads). A total of 20 µL of the SP3 beads were added to the alkylated sample, followed by another 2.5 times the sample liquid volume of ethanol, and the solution was mixed at room temperature for 20 minutes. After washing the beads twice with 80% ethanol, 100 µL of 50 mM Tris-HCL pH 8.0 was added and mixed. A total of 500 ng of Trypsin/Lys-C Mix (Promega K. K, Tokyo, Japan) was added for peptide fragmentation and incubated at 37°C overnight. Next, 5% TFA (trifluoroacetic acid)(20 µL) was added, and the sample was processed in a sample-sealed ultrasonic disruption machine. The sample was then desalted using a reversed-phase spin column (GL-Tip SDD; GL Sciences, Tokyo, Japan) and dried using a centrifugal evaporator. To this, 2% ACN-0.1% TFA was added, and the peptides were dissolved using a sample-sealed sonication system. The peptide concentration was measured by a BCA assay and adjusted with 2% ACN-0.1% TFA to reach a peptide concentration of 200 ng/µL. Finally, an LC-MS analysis was conducted.
NanoLC-MS analyses
The analysis was carried out as a multi-omics analysis work provided by Promega K.K. (Tokyo, Japan). Injected peptide volume: 300 ng, nanoLC used: UltiMate 3000 RSLCnano LC System (Thermo Fisher Scientific, K.K.), column size: 75 um i.d. × 120 mm length (Nikkyo Technos, Co., Ltd, Tokyo, Japan), column temperature: 40°C, Solvents: [A], distilled water with 0.1% formic acid, [B] 80% ACN with 0.1% formic acid. Scaffold DIA (Proteome Software, Inc, Portland, OR, USA) was used for the data analysis. MS analysis conditions, MS: Q Exactive HF-X (Thermo Fisher Scientific, K.K.). Ionization method: ESI positive mode, time: 80 min (gradient time of 4–84 min measured). MS analysis: Overlapping window DIA. Scaffold DIA (Proteome Software) was used for the data analysis. Protein Sequence Database: Human UniProtKB/Swiss-Prot database (Proteome ID UP000005640). The Protein Sequence Database was: Human UniProtKB/Swiss-Prot database (Proteome ID UP000005640).
Data analyses using the Genome Enhancer (geneXplain GmbH, Wolfenbüttel, Germany)
The analysis was carried out as a multi-omics analysis work provided by Promega K.K. (Tokyo, Japan). The Genome Enhancer release 3.0 (TRANSFAC® and TRANSPATH® release 2022.1) platform was used for the data analysis. TRANSFAC® is a database of TF binding sites, used to identify TFs that regulate expressed or mutated genes. TRANSPATH® is a database of mammalian signaling and metabolic pathways that searches for general master regulators of TFs identified by TRANSFAC®.
This approach comprises two major steps: (1) analyzing promoters and enhancers of DEGs for the TFs involved in their regulation, rendering them important for the process under study; and (2) re-constructing the signaling pathways that activate these TFs and identifying master regulators at the top of such pathways. For the first step, the TRANSFAC®68 database is employed, together with the TF binding site identification algorithms Match69 and CMA52. The second step involves the signal transduction database TRANSPATH®53 and special graph search algorithms70 implemented in the "Genome Enhancer" software program.
Databases used in the study of TRANSFAC®
The analysis was carried out as a multi-omics analysis work provided by Promega K.K. (Tokyo, Japan). Transcription factor binding sites in promoters and enhancers of DEGs were analyzed using known DNA-binding motifs described in the TRANSFAC® library, release 2022.1 (geneXplain GmbH) (https://genexplain.com/transfac). The master regulator search uses the TRANSPATH® database (BIOBASE), release 2022.1 (geneXplain GmbH) (https://genexplain.com/transpath). A comprehensive signal transduction network of human cells is built by the software program based on the reactions annotated in TRANSPATH®.
Methods for analyzing enriched TF binding sites and composite modules of TRANSFAC®
The analysis was carried out as a multi-omics analysis work provided by Promega K.K. (Tokyo, Japan). Transcription factor binding sites in promoters and enhancers of DEGs were analyzed using known DNA-binding motifs. The motifs are specified using PWMs that assign weights to each nucleotide in each position of the DNA binding motif for a TF or a group of them. We search for TFBS that are enriched in the promoters and enhancers being studied compared to a background sequence set, such as promoters of genes that were not differentially regulated under the experimental condition. We denote “study” and “background” sets briefly as “Yes” and “No” sets.
In the present study, we used a workflow considering promoter sequences of a standard length of 1100 bp (-1000 to + 100). The error rate in this part of the pipeline is controlled by estimating the adjusted p-value (using the Benjamini-Hochberg procedure) in comparison to the TFBS frequency found in randomly selected regions of the human genome (adjusted p-value < 0.01). We used the CMA algorithm to search for composite modules69 in the promoters and enhancers of the Yes and No sets. We searched for a composite module consisting of a cluster of 10 TFs in a sliding window of 200–300 bp that significantly separates sequences in the Yes and No sets (minimizing Wilcoxon p-value).
Methods for identifying master regulators in networks of TRANSFAC®
The analysis was carried out as a multi-omics analysis work provided by Promega K.K. (Tokyo, Japan). We searched for master regulator molecules in signal transduction pathways upstream of the identified TFs. The master regulator search uses a comprehensive signal transduction network of human cells. The main algorithm of the master regulator search has already been described71,72. The goal of the algorithm is to find nodes in the global signal transduction network that may potentially regulate the activity of a set of TFs found in a previous step in the analysis. Such nodes are considered the most promising drug targets, since any influence on such a node may switch the transcriptional programs of hundreds of genes that are regulated by the respective TFs. In our analysis, we ran the algorithm with a maximum radius of 12 steps upstream of each TF in the input set. The error rate of this algorithm is controlled by applying it 10000 times to randomly generated sets of input TFs of the same set size. The Z-score and FDR (false discovery rate) value of ranks are then calculated for each potential master regulator node on the basis of these random runs53. We control the error rate with an FDR threshold of 0.05.
Microarray analyses
The measurement was outsourced to DNA Chip Research Inc. (Tokyo, Japan), and the Agilent SurePrint G3 Human GE 8x60K Microarray (Design ID 028004) was used for the analysis. The data were subjected to a heatmap analysis, cluster analysis, and principal component analysis. Human ES cells and the human iPSC line N116213 from GEO data (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE43328) 73 were used as controls.
Data Availability
The datasets generated and/or analysed during the current study are available in the GSE230084 repository, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE230084).
Shotgun LC-MS/MS
The experiment was conducted by Apro Science Group / Pharma Foods International Co.,Ltd (Tokushima, Japan). We used an EzRIPA Lysis kit (ATTO Corporation, Tokyo, Japan) for cell lysis according to the manufacturer’s instructions. A protein solution of 2362 µg/mL was obtained from the concentrated solution of the culture supernatant of MEFs. Finally, 2.0 µg of protein was used for LC–MS/MS (EASY-nLC 1200 System; Thermo Fisher Scientific K.K.). The samples were analyzed with a Nanoflow-LC ESI using an Q Exactive plus (Thermo Fisher Scientific K.K.) at the Pharma Foods International Co., Ltd. (Tokushima, Japan). Based on the protein quantification findings, Tris buffer containing Dithiothreitol was added to 2 µg of sample to achieve a final concentration of 5 mM, and the sample was reduced at 35°C for 2 h. To the reduced sample, we further added Tris buffer containing Iodoacetamide to a final concentration of 14 mM at room temperature for 30 minutes (light shielded), followed by Trypsin. The collected sample solution was solvent-substituted on a cation exchange column (EASY-Spray column, 15 cm × 75 µm ID, 3 µm particles, 100 Å pore size; Thermo Fisher Scientific K.K.), desalted and concentrated, and a portion was subjected to LC-MS/MS. Solvent A was 0.1% formic acid, and Solvent B was 80% acetonitrile/0.1% formic acid. The peptides were eluted in a 229-min gradient of 5% solvent B in solvent A to 40% solvent B in solvent A at 300 nL/min. In-house database searches were performed using the MASCOT Server (http://www.matrixscience.com/help_index.html) with product ion measurement data for all MS/MS spectra obtained from LC-MS/MS.
Cell differentiation assays
To functionally verify the ability of a hiPSC to differentiate into three germ layers (ectoderm, mesoderm, and endoderm), the STEMdiff Trilineage differentiation kit (Stemcell Technologies Inc., Vancouver, Canada) was used.
qPCR array
RNA was prepared using a SuperPREP II Cell Lysis & RT Kit for qPCR (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer's instructions. Real-time PCR was performed using a StepOnePlus system (Life Technologies, Carlsbad, CA, USA). Luna Universal qPCR Master Mix (New England Biolabs Inc., Ipswich, MA, USA) was used according to the manufacturer's instructions74,75. The PCR protocol was as follows: (1) initial denaturation at 95°C for 10 min; (2) denaturation at 95°C for 15 sec; continued denaturation of the double-stranded DNA; (3) annealing of primers at 60°C for 60 sec, repeat steps (2)-(3) 40 times; (4) denaturation at 95°C for 15 sec, annealing of primers at 60°C for 60 sec, and denaturation at 95°C for 15 sec [Melt Curve Stage].
For the mRNA expression analysis, a TaqMan Array 96-Well FAST Plate (Human Stem Cell Pluripotency or Human DNA Repair Mechanism; Applied Biosystems) was used with TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific, K.K.) according to the manufacturer's instructions. The PCR protocol was as follows: (1) denature at 95°C for 20 sec, continue denaturing the double-stranded DNA; (2) anneal primers at 60°C for 20 sec, and repeat steps (1)-(2) 40 times.
For the analysis of real-time PCR data using a TaqMan™ Array96-Well FAST Plate, 18S, GAPDH, HPRT1, and GUSB were used as housekeeping genes. The maximum CT value was set at 40. The ΔCT for undetected was calculated by subtracting the average of the CT values of the 4 housekeeping genes from the maximum CT value (40). The ΔCT value of the target was calculated by subtracting the average CT values of the four housekeeping genes from the CT values of the various genes under each culture condition. To calculate the ΔΔCT values of the target, the average ΔCT values of the various genes under control culture conditions were subtracted from the ΔCT values of the various genes under each culture condition. ΔΔCT values were then calculated using the Excel software program (Microsoft Corporation, Redmond, WA, USA). Primers were purchased from Takara Bio (Shiga, Japan), as follows: Homo sapiens actin beta (ACTB), NM_001101.5 (HA067803; Takara); Homo sapiens PR/SET domain 13 (PRDM13), NM_021620.4 (HA351448; Takara); Homo sapiens PR/SET domain 16 (PRDM16), transcript variant 1, NM_022114.4 (HA250465; Takara); Homo sapiens PR/SET domain 15 (PRDM15), transcript variant 1, NM_022115.7 (HA326903; Takara); Homo sapiens PR/SET domain 14 (PRDM14), NM_024504.4 (HA168102; Takara); Homo sapiens nuclear receptor binding SET domain protein 1 (NSD1), transcript variant 3, NM_001365684.1 (HA392808; Takara); Homo sapiens ribosomal oxygenase 1 (RIOX1), NM_024644.5 (HA326739; Takara); Homo sapiens suppressor of variegation 3–9 homolog 2 (SUV39H2), transcript variant 1, NM_001193424.2 (HA197608; Takara); Homo sapiens zinc finger protein 408 (ZNF408), transcript variant 2, NM_001184751.2 (HA317725; Takara); Homo sapiens euchromatic histone lysine methyltransferase 1 (EHMT1), transcript variant 2, NM_001145527.2 (HA165906; Takara); Homo sapiens lysine demethylase 7A (KDM7A), NM_030647.2 (HA145925; Takara); Homo sapiens SET domain containing 7, histone lysine methyltransferase (SETD7), NM_001306199.2 (HA380534; Takara); Homo sapiens SET domain containing 3, actin histidine methyltransferase (SETD3), NM_032233.3 (HA361378; Takara); Homo sapiens DOT1 like histone lysine methyltransferase (DOT1L), NM_032482.3 (HA391440; Takara); Homo sapiens lysine methyltransferase 5C (KMT5C), NM_032701.4 (HA341823; Takara); Homo sapiens PR/SET domain 6 (PRDM6), NM_001136239.4 (HA325705; Takara); Homo sapiens SET and MYND domain containing 1 (SMYD1), NM_001330364.2 (HA348945; Takara); Homo sapiens jumonji domain containing 1C (JMJD1C), transcript variant 3, NM_001282948.2 (HA359298; Takara); Homo sapiens lysine demethylase 1B (KDM1B), transcript variant 1, NM_001364614.2 (HA381585; Takara); Homo sapiens lysine demethylase 4E (KDM4E), NM_001161630.1 (HA354929; Takara); Homo sapiens lysine demethylase 3B (KDM3B), NM_016604.4 (HA309120; Takara); Homo sapiens nuclear receptor binding SET domain protein 3 (NSD3), NM_017778.3 (HA364537; Takara); Homo sapiens lysine demethylase 4D (KDM4D), NM_018039.3 (HA375920; Takara), Homo sapiens lysine demethylase 3A (KDM3A), transcript variant 2, NM_001146688.2 (HA363808; Takara); Homo sapiens ASH1 like histone lysine methyltransferase (ASH1L), transcript variant 1, NM_001366177.2 (HA339441; Takara); Homo sapiens SET and MYND domain containing 2 (SMYD2), NM_020197.3 (HA357766; Takara); Homo sapiens PR/SET domain 8 (PRDM8), transcript variant 2, NM_001099403.2 (HA168010; Takara); Homo sapiens PR/SET domain 9 (PRDM9), transcript variant A, NM_001310214.2 (HA361223; Takara); Homo sapiens PR/SET domain 10 (PRDM10), transcript variant 10, NM_001367890.1 (HA322746; Takara); Homo sapiens PR/SET domain 11 (PRDM11), transcript variant 1, NM_001256695.2 (HA355454; Takara); Homo sapiens lysine methyltransferase 2C (KMT2C), NM_170606.3 (HA115614; Takara); and Homo sapiens PR/SET domain 12 (PRDM12), NM_021619.3 (HA375190, Takara).
Cell proliferation assays
Cell proliferation was measured using a Countess (Thermo Fisher Scientific K.K.).
Analysis of binding proteins
The experiment was conducted by IDEA Consultants, Inc. (Tokyo, Japan).
(1) Preparation of His-tag protein-anchored beads
Binding and washing buffer (50 mM sodium phosphate [pH 8.0], 300 mM NaCl, 0.01% Tween-20). A total of 50 µg of binding protein (Recombinant Human MFG-E8 [His-tag]) was dissolved in 700 µL of binding buffer. Approximately 20 µL was saved for final SDS-PAGE. Magnetic beads (Dynabeads His-Tag Isolation & Pulldown 2 mg (50 µL) supernatant removed after 2 min of magnet fixation. Incubations were performed for 10 min at room temperature using a rotator. Fixation and washing (300 µL of washing solution was used four times for 2 min of magnetic fixation) were performed After the binding of MFG-E8 to the beads, the protein in the supernatant was quantified by Nano Drop and the amount of MFG-E8 protein fixed on the beads was estimated from the decrease in the amount.
(2) The pull-down analysis
Pull-down buffer (6.5 mM sodium phosphate [pH 7.4], 70 mM NaCl 0.01% Tween-20). Wash buffer (50 mM Sodium phosphate [pH 8.0], 300 mM NaCl, 0.01% Tween-20). Elution buffer (300 mM Imidazole, 50 mM sodium phosphate [pH 8.0], 300 mM NaCl, 0.01% Tween-20). Magnetic beads. (1) His-tag protein-immobilized magnetic beads 2 mg. (2) 2 mg of unfixed magnetic beads (negacon). Protein solution (200 µL of sent protein + 1.8 mL pull-down buffer (dilute 10-fold). Two portions of His-tag protein-anchored and unfixed beads use 1 mL of each. Incubation (4°C for 2 h using a rotator). Fixation and washing (300 µL of washing buffer 4 times with 2 min of magnet fixation). Elution (elution buffer 50 µL, rotator 5 min at room temperature, supernatant obtained after magnet fixation for 2 min). Two batches of 1 mL each of His-tag protein-immobilized and non-immobilized beads were used. Incubations were carried out for 2 h at 4°C using a rotator.
Fixation and washing (300 µL of wash buffer four times, 2 min magnetic fixation) were performed. Elution (50 µL elution buffer, rotator 5 min, room temperature, supernatant obtained after 2 min of magnet fixation) was performed.
(3) SDS-PAGE
The elution sample consisted of 30 µL elution sample + 10 µL 4 × sample buffer, with 3 lanes of each sample (5 µL, 10 µL, and 20 µL/lane). For SDS-PAGE gels, e. Pagel (5–20%) was used.
For staining: silver staining (without glutaraldehyde) was used.
The ToxTracker assay
Experiments and analyses were carried out by Toxys (Oegstgeest, Netherlands). The ToxTracker assay was performed by Toxys (Oegstgeest, Netherlands). ToxTracker reporter cells were maintained by culturing them in gelatin-coated dishes in the presence of irradiated primary MEFs in mES cell culture medium. During chemical exposure and reporter analyses, the ToxTracker cells were cultured in the absence of fibroblasts in mES cell (strain B4418) culture medium.
For cytotoxicity testing/dose range finding, wild-type mES cells were exposed to 20 different concentrations of the test substances, with a maximum concentration of 6 µg/ml of MFG-E8. Cytotoxicity was estimated by the cell count after 24-h exposure using a flow cytometer and was expressed as the percentage of viable cells after 24-h exposure compared to vehicle-exposed controls.
For the ToxTracker assay, 6 independent mES reporter cell lines were seeded in 96-well gelatin-coated cell culture plates in 200 µl of mES cell medium (50,000 cells/well). At 24 h after seeding the cells in the 96-well plates, the medium was aspirated, and fresh mES cell medium containing 10% fetal calf serum and the diluted chemicals was added to the cells. For the tested materials, five concentrations were tested in two-fold dilutions. The induction of the GFP reporters was determined after 24-h exposure using a flow cytometer. Only the GFP expression in intact single cells was determined. The mean GFP fluorescence was measured to calculate the GFP reporter induction compared to vehicle control treatment. Cytotoxicity was estimated by the cell count after 24-h exposure using a flow cytometer and was expressed as percentage of intact cells after 24-h exposure compared to vehicle-exposed controls.
For the cytotoxicity assessment in the ToxTracker assay, the relative cell survival for the six different reporter cell lines was averaged, as the cytotoxicity levels were similar. Metabolic activation was included in the ToxTracker assay by addition of S9 liver extract from Phenobarbital/5,6-Benzoflavone-induced rats (Moltox). Cells were exposed to 5 concentrations of the test samples in the presence of 0.4% S9 and required co-factors (RegenSysA + B, Moltox) for 24 h. Positive reference treatments with cisplatin (DNA damage), diethyl maleate (oxidative stress), tunicamycin (unfolded protein response), and cyclophosphamide (metabolic activation of progenotoxins by S9) were included in all experiments. The solvent concentration was the same in all wells and never exceeded 1% for DMSO. If auto-fluorescence of the test substance was observed in the dose range finding, wild-type mES cells were exposed to the test samples at the same concentrations as that used in the ToxTracker assay. The mean fluorescence caused by the sample was then subtracted from the ToxTracker results for the sample.
Activation of the Bscl2-GFP or Rtkn-GFP reporters indicates the induction of DNA damage, activation of Srxn1-GFP and Blvrb-GFP indicates the induction of cellular oxidative stress, and activation of Ddit3-GFP indicates the unfolded protein response. The Btg2-GFP reporter is controlled by the p53 tumor suppressor and activated by DNA damage but can also be induced by oxidative stress, hypoxia, metabolic stress, and apoptosis. The ToxTracker assay is thus considered to have a positive response when a compound induces at least a two-fold increase in the GFP expression in any of the reporters at one or more test concentrations.
To allow for the comparison of induction levels of the ToxTracker reporter cell lines for a large number of compounds, the makers developed Toxplot, a dedicated data analysis software package. Toxplot imports raw GFP reporter data from the flow cytometer, calculates GFP induction levels and cytotoxicity, performs statistical analyses of the data and hierarchical clustering of the tested compounds, and visualizes the data in a heatmap, allowing for the convenient interpretation of obtained test results. The ToxPlot software program uses agglomerative hierarchical clustering to visualize the ToxTracker data, calculating for each compound the level of GFP induction for every individual reporter at a specified level of cytotoxicity (typically 10%, 25%, 50%, and 75%). The GFP induction levels are calculated by linear regression between two data points around the specified cytotoxicity level. If the specified level of cytotoxicity cannot be reached at the highest tested compound concentration, Toxplot displays the GFP induction level at this top concentration. In the heatmap, Toxplot clearly marks the compounds that do not induce the selected level of cytotoxicity. Because the cytotoxicity for a compound can vary between the ToxTracker cell lines, calculations of the GFP induction levels of the individual reporters by Toxplot can slightly deviate from the GFP induction and cytotoxicity Figs. 56.
Statistical analyses
Comparisons between multiple groups (more than two groups) were performed using a one-way analysis of variance with the StatPlus software program (AnalystSoft, Walnut, CA, USA). Statistical significance was set at *P < 0.05 or **P < 0.01 for all tests.