br Equal amounts RNA input
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.
2.5. Gene silencing of METTL3 and Gb3 synthase
Silencing of METTL3 and globotriaosylceramide (Gb3) synthase was accomplished as described previously [27,32,36]. siRNA-METTL3 (si-METTL3, 100 nM) or siRNA-Gb3 synthase (siGb3S) and siRNA-scram-bled control (siRNA-SC; 100 nM) were introduced into TP53-Dox Concanamycin-A (3 × 106/100-mm dish; 4000 cells/well in 96-well plates) after over-night growth, facilitating with Lipofectamine 2000 in Opti-MEM re-duced-serum medium (Thermo Fisher Scientific) for 4 h. The cells continuously grew in 5% FBS medium for an additional 48 h. In com-bination treatment groups, cells were then treated with Dox (5 µM) in 5% FBS medium for an additional 48 h. Biochemical Pharmacology 160 (2019) 134–145
Under the various treatment conditions, cells (50,000 cells/chamber) were grown in 4-chamber slides for 48 h. After methanol fixation, cells were blocked with 5% goat serum in phosphate-buffered saline (PBS), and incubated with antibodies against METTL3 (1:2000 dilution) and phosphorylated p53 Ser15 (1:200 dilution) in blocking solution at 4 °C, overnight. Primary antibodies were further recognized by Qdot 605- or Alexa Fluor 488-conjugated goat IgG (1:50 or 1:2000, respectively). Cell nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole) in mounting solution (Vector Laboratories, Burlingame, CA, USA). Images (100× magnification) were captured using an Olympus BX63 automated fluorescence microscope with monochrome CCD camera (Olympus, Tokyo, Japan). Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) and Qdot 605-conjugated goat anti-rabbit IgG (H + L) sec-ondary antibodies were purchased from Thermo Fisher Scientific.
2.7. Western blotting analysis
Western blotting was carried out as described previously [27,37]. Briefly, cells or tissue homogenates were lysed in NP40 cell lysis buffer (Biosource, Camarillo, CA, USA) to extract total cellular proteins once a given treatment was finished. Detergent-soluble proteins were mea-sured by using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of proteins (50 or 100 µg/lane) were resolved by using 4–20% gradient SDS-PAGE (Thermo Fisher Scien-tific). After transferring, blots of nitrocellulose membrane were blocked in 5% fat-free milk in 0.05% Tween-20, 20 mM phosphate-buffered saline, pH 7.4 (PBST), and then incubated separately with each one of the primary antibodies (1:500 or 1:2000 dilution), at 4 °C overnight. After PBS washing, these blots were incubated with corresponding horseradish peroxidase conjugated secondary antibodies (1:5000 dilu-tions) and developed using SuperSignal West Femto substrate (Thermo Fisher Scientific). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control for cellular protein. Relative protein levels present were calculated from the optical density (OD) values of bands in triplicated blotting, normalized against corre-sponding GAPDH OD values in the same membranes. Antibodies against human p53 phosphorylated at Ser15 or for poly(ADP-ribose) polymerase (PARP; Cat. # 9542S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody for methyltransferase-like 3 (METTL3) was purchased from Thermo Fisher Scientific. Antibodies for PUMA, p21, Bax, p53, β-catenin, phosphorylated cSrc and GAPDH were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).