Fig 1.
Asymmetric total synthesis of (+)-Anti-mefloquine [(+)-1] and (–)-anti-mefloquine [(–)-2].
Fig 2.
Identification of mefloquine as a potent enhancer of G418-induced TP53 PTC readthrough.
(A) Automated fluorescence microscopy analysis of PTC readthrough. HDQ-P1 cells in 96-well plates were treated for 48 h with 20 μM of the indicated drugs in the presence or absence of 50 μM G418 and nuclear p53 levels were measured. Data are expressed as the mean ± SD (n = 6 biological replicates for controls, n = 3 biological replicates for drug treatments) and were collected from two independent experiments, represented by the break in the graph. (B) Representative images of p53 immunofluorescence from A. Blue shows DNA staining and green is p53 immunofluorescence (scale bar, 100 μm). (C) Western analysis of p53 expression following 48 h treatment. Samples were analysed by automated quantitative capillary electrophoresis western analysis with a p53 antibody to measure full-length p53 (FL-p53) and truncated p53 (TR-p53), and a vinculin antibody. The chemiluminescence signals are shown as pseudo-blots. The amount of FL-p53 was normalized to that of vinculin. FL:FL represent the ratio of FL-p53 in treated samples to FL-p53 in cells exposed to G418 alone. (D) Western analysis of p53 expression over time. Samples were analysed by automated quantitative capillary electrophoresis western analysis as above.
Fig 3.
PTC readthrough enhancement by mefloquine and individual stereoisomers in HDQ-P1 cells after 48 h exposure (A) and in human cancer cell lines harboring different nonsense mutations in TP53 after 72 h exposure (B-D). FL-p53 was measured and expressed as in Fig 1.
Fig 4.
Effect of ionizing radiation on the Ser15 phosphorylation of p53 produced by PTC readthrough.
The indicated cancer cells were treated with compounds for 48 h and exposed to 4 Gy ionizing radiation 24 h prior to harvesting. Following treatment, p53 and phosphorylated p53 (pp53 Ser15) levels were measured by automated capillary electrophoresis western analysis. Vinculin and FL-53 were measured in the same capillary while pp53 (Ser15) was measured in a duplicate capillary, indicated by the box. Numbers for FL-p53 and pp53 are chemiluminescence values normalized to that of vinculin and divided by 1000.
Fig 5.
Effect of mefloquine, G418 and irradiation on TP53 and P21 expression.
Cells were treated as indicated and relative TP53 and P21 transcript levels were quantified by RT-qPCR. Data are expressed as the mean ± SD (n = 3 biological replicates). GAPDH served as the reference gene.
Fig 6.
(A) G418 was added with or without mefloquine at t = 0 and its total intracellular levels were measured at the indicated times. The solid line shows logarithmic curve fit. (B) Cells were exposed or not to G418 and mefloquine for 24 h and subjected to crude subcellular fractionation to determine cytosolic and organellar G418 concentrations, as well as total cellular G418 concentration. Western analysis shows the amount of TPP1, a lysosomal protein, and GAPDH, a cytosolic protein, in each fraction. Data are expressed as the mean ± SD (n = 3 biological replicates).