br Cell viability assay br Cell viability
2.2. Cell viability assay
Cell viability was determined by quantitation of ATP, an indicator of live cells, using the CellTiter-Glo luminescent cell viability assay kit (Promega, Madison, WI, USA), as described previously [19,32]. Briefly, SB 203580 (4000 cells/well) were grown in 96-well plates with 10% FBS supplemented RPMI-1640 medium. Cells were treated with indicated agents in 5% FBS medium for 72 h. For combination treatments, cells (3 × 106/100-mm dish; 4000 cells/well in 96-well plates) were pre-treated with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-pro-panol (PDMP; 5 µM) or neplanocin A (NPC, 20 nM), fumonisin B1 (FB1, 25 µM), Shiga toxin 1B-subunit (STxB, 100 nM), PP2 (500 nM) and FH535 (5 µM) in 5% FBS medium for 48 h. ATP quantitation for cell viability assessment was accomplished with a Synergy HT microplate reader (BioTek, Winnooski, VT, USA), following incubation with Cell-Titer-Glo reagent.
2.3. Induction and detection of apoptosis
Apoptosis was carried out using an APO-DIRECT kit (BD Biosciences, San Jose, CA, USA) and propidium iodide (PI), as described previously [32,33]. Briefly, cells were pretreated with NPC (20 nM) or vehicle in 5% FBS medium for 3 days, and then co-treated with NPC and
Dox (100 nM) for an additional 48 h to induce apoptosis. After fixing with paraformaldehyde (1% w/v) and 70% ethanol, cells (1 × 106 cells/ml) were incubated with FITC-labeled Br-dUTP (5 μg/ 100 μl) and then PI (5 μg/100 μl) in staining buffer containing RNase for apoptosis detection. Cells were then analyzed using an imaging flow cytometry system, namely an ImageStreamX Mark II with high-resolu-tion microscope (Amnis, Seattle, WA, USA). For each sample, 5000 events were counted in triplicate. Data were analyzed with the IDEAS® Software package (Amnis).
2.4. N6-Methyladenosine RNA immunoprecipitation (MeRIP) and RT-qPCR
After treatments, total RNA was extracted from cells of SW48-Dox, TP53-Dox and WiDr cell lines using SV total RNA isolation kits (Promega). MeRIP was performed using the Magna MeRIP m6A kit (Cat.
# 17-10499, EMD Millipore, Billerica, MA, USA), as described in pre-vious studies [28,34] and per kit instructions. Briefly, equal amounts of total RNA (300 µg in 1 µg/µl aliquots) were fragmented with heating in a thermocycler, extracted, and validated with 1.5% agarose gel elec-trophoresis, and then quantitated via NanoDrop spectrometry. Frag-mented RNA samples (∼100 nt, 280 µg) were incubated with anti-m6A antibody (clone 17-3-4-1; 10 µg) bound-protein A/G magnetic beads in IP buffer for 2 h at 4 °C, eluted with elution buffer, and purified in RNeasy mini-columns. For control experiments, MeRIP was carried out with mouse IgG replacing m6A antibody, under the same conditions.
Equal amounts RNA input (fragmented RNA; 100 ng) and MeRIP RNA (equal to 100 ng input) from cell extract samples were amplified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) with the QuantiTect SYBR Green RT-PCR kit (Qiagen, Germantown, MD, USA), coupling to the MyiQ Real-Time PCR detec-tion system, as described previously . To detect the m6A at codon 273 of p53, a 95-bp fragment in the region of human TP53 mRNA (codons 249–280; accession number BC003596.1) was generated by using the upstream primer (5′-AGGCCCATCCTCACCATCAT-3′) and downstream primer (5′-CTCCCAGGACAGGCACAAAC-3′). This 95-bp product that includes the point-mutation site (codon 273) would be transited and methylated in p53 R273H mutants. MeRIP-positive con-trol RNA was generated using MeRIP primers human EEF1A1 (acces-sion# NM_001402) positive: forward primer (5′-CGGTCTCAGAACTGT TTGTTTC-3′) and reverse primer (5′-AAACCAAAGTGGTCCACAAA-3′) to amplify the methylated region (stop codon); MeRIP-negative control RNA was generated using MeRIP primers human EEF1A1 negative: forward primer (5′-GGATGGAAAGTCACCCGTAAG-3′) and reverse primer (5′-TTGTCAGTTGGACGAGTTGG-3′) to amplify the un-methylated region (exon 5). A standard curve was generated using threshold cycle (Ct) values of serially diluted input RNA (fragmented) from SW48-Dox cells, calculating the fold enrichment as the ratio of Cm6A to Cnegativecontrol. During RT-PCR reactions, cDNA synthesis was conducted at 50 °C for 10 min; polymerase was then inactivated at 95 °C for 1 min, and 40 cycles were run from denature (95 °C, 10 s) to anneal (60 °C, 30 s). All samples were analyzed in triplicate, repeated at least two times.