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  • br Immunoprecipitation and protein identification br

    2020-08-12


    2.8. Immunoprecipitation and protein identification
    After treatments, p53 proteins were precipitated with anti-p53 or anti-phosphorylated-p53 Concanamycin-A and identified by mass spectro-metry (MS), accomplished as described previously [38]. In-gel trypsin digestion and tandem mass spectrometry (MS/MS) analysis of SDS-PAGE protein bands was conducted at the Proteomics Core at Uni-versity of Arkansas for Medical Sciences. The Mascot database was drawn on for protein identification, which was accomplished using Scaffold software (v. 4.8, Proteome Software, Portland, OR, USA).
    2.9. Chemicals and statistical analysis
    All chemicals used in this study were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. PDMP was purchased from Matreya (State College, PA, USA). (−)-Neplanocin A (NPC), which was demonstrated to block m6A formation by acting as a selec-tive inhibitor of S-adenosylhomocysteine (SAH) hydrolase and thereby act as an indirect inhibitor of METTL3 activity (due to excess SAH ac-cumulation) [39], was purchased from Cayman Chemical (Ann Arbor, MI, USA). Fumonisin B1 (FB1), a ceramide synthase inhibitor [27], was
    RNA: AUG GAG GAG UUU GAG GUG CGU GUU UGU GCC UGU CCU
    RNA: AUG GAG GAG UUU GAG GUG CAU GUU UGU GCC UGU CCU
    Cell viability (% of control) 
    TP53-Dox vehicle C.
    Vehicle siRNA-SC
    TP53-Dox siMETTL3
    700 NPC siMETTL3
    PDMP *
    SW48-Dox vehicle
    Doxorubicin [µM]
    Cell Responses to Treatments
    purchased from Biomol (Plymouth Meeting, PA, USA). A cSrc kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)-pyrazolo[3,4-d]pyr-imidine (PP2) [40] was purchased from Enzo Life Sciences (Plymouth Meeting, PA, USA). An effective β-catenin/Tcf inhibitor, FH535 (2,5-dichloro-N-(2-methyl-4-nitrophenyl)-benzenesulfonamide) [41], was purchased from Sigma-Aldrich. Shiga toxin 1B-subunit (STxB) was kindly provided by Dr. Anne V. Lane (Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, and Tufts University School of Medicine, Boston, MA) [42]. siRNAs targeting human METTL3 (sc-92172) [29], Gb3 synthase (sc-72831) [36] and the scrambled control (siRNA-SC, sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
    All experiments were repeated at least 2 times. All data were ana-lyzed using the GraphPad Prism software (GraphPad, San Diego, CA, USA). Statistical analyses, which included the two-tailed Student’s t-test or ANOVA, were used to compare the continuous variables between groups. Statistical significance was considered for p < 0.05.
    3. Results
    3.1. p53 R273H with RNA m6A methylation correlates to cancer drug resistance 
    Table 1
    Cell Lines and Doxorubicin (nM) Oxaliplatin (µM) 5-Fluorouracil (µM)
    Pretreatments
    The R273H mutation of TP53 is the most common one detected, accounting for approximately 3.1% of p53 mutant-associated cancer cases [11]. p53 R273H protein is encoded from mutant mRNA, in which CAU replaces CGU at codon 273 (Fig. 1A). Colon cancer TP53-Dox or TP53-5FU cells, which heterozygously carried R273H mutant p53 (mut p53, introduced by a CRISPR/Cas9 system in SW48 cells) and were long-term exposed to low-dose doxorubicin (Dox) or 5-flurouracil (5-FU), displayed multidrug resistance, as compared with SW48-Dox or 
    SW48-5FU cells, which were cells that were comparably long-term exposed to low concentration Dox or 5-FU, but which carry only wt p53. To wit, in TP53-Dox cells, the IC50 values for Dox increased by ten-fold, and for oxaliplatin (Oxa) or 5-FU, increased by two-fold (Fig. 1B, C, Table 1), respectively, over the IC50 values observed for SW48-Dox cells that were subjected to the same treatments. In subline TP53-5FU,
    Area Thresholdreshold30%30%PI PI Nonapoptotic
    BrdU
    BF
    PI
    BrdU
    BF
    PI
    TP53-Dox cells
    ApoptoticTP53-Doxcells
    FITC BRDU
    ity FITC BRDU
    ity FITC BRDU
    FITC BRDU
    Intensity BrdU
    Intensity BrdU
    Intensity BrdU
    Intensity BrdU
    Apoptotic (% of total cells) 
    Vehicle C. SW48-Dox TP53-Dox cellsp
    NPC PARP Vehicle NPC
    Vehicle NPC 116 kDa
    Cleaved-P
    GAPDH
    Fig. 2. Inhibition of m6A methylation increased Dox-induced apoptosis in TP53-Dox cancer cells. SW48-Dox and TP53-Dox cells were treated with neplanocin A (NPC, 20 nM) or vehicle for 3 days, and then exposed to Dox (100 nM, for 48 h) with these treatments to induce apoptosis. Imaging flow cytometry was accomplished with an ImageStream X Mark II system, and the data analyzed with IDEAS® Software. A, Apoptosis assay with imaging flow cytometry. BrdU, bromodeoxyuridine-FITC; BF, bright field; PI, propidium iodide. Top-panel, images of apoptotic and nonapoptotic cells of TP53-Dox cells identified using imaging flow cytometry for analysis. Apoptotic cells labelled with BrdU-FITC (green) are presented as percentages of total cells detected in each sample. B, NPC effects on apoptosis. **, p < 0.001 compared to SW48-Dox cells; *, p < 0.001 compared to cells treated with vehicle. C, Representative Western blot of PARP protein. After above treat-ments, equal amounts of detergent-soluble proteins extracted (50 µg/lane) were resolved using 4–20% gradient SDS-PAGE, and then immunoblotted with PARP antibody. Cleaved-P, cleaved PARP; GAPDH served as internal control. Protein levels (bottom panel) are represented as means of their optical densities normalized against GAPDH from three settings of blots. *, p < 0.01 compared to corresponding cells treated with vehicle.