1. Overexpression of Pim kinases contributes to increased protein translation in resistant cells through controlling cap-independent translation.
Overexpression of Pim kinases contributes to increased protein translation in resistant cells through controlling cap-independent translation. A, de novo protein synthesis was measured by 35S-methionine incorporation and normalized to total protein levels in EBC-1 and its resistant subline EBC-1PHAR treated with PHA (PHA; 0.1 μmol/L), LGB321 (LGB; 1 μmol/L), or BEZ235 (0.5 μmol/L) for 3 hours. Results shown are the mean ± SD from three experiments, each group in two replicates. B, autoradiography of lysates from A after the SDS-PAGE. Coomassie blue staining of total protein is shown. C, global RNA synthesis was measured by the incorporation of the uridine analogue EU into newly transcribed RNA in EBC-1 and its resistant subline PHAR. The modified RNA was detected with Alexa Fluor 488 azide (green fluorescence). Hoechst dye was used as a nuclear counterstain (blue fluorescence). EBC-1 cells were treated with actinomycin D (ActD; 5 μg/mL) for 3 hours. NL, no EU labeling in EBC-1 cells. Representative images are shown. Scale bar, 100 μmol/L. D, The fluorescent signal intensities in C and Supplementary Fig. S6C were quantified by normalizing the intensity of green signal to that of the blue signal using the ImageJ software. Results shown are the mean ± SD from three experiments, each group in two replicates. E and F, luciferase activities were measured in cells transfected with an HCV IRES or Bcl-2 IRES plasmid and treated with LGB321 (LGB; 1 μmol/L), BEZ235 (0.5 μmol/L), or PP242 (1 μmol/L). The relative ratio of firefly/Renilla luciferase activity are shown. Results shown are the mean ± SD from three experiments, each group in three replicates. G, m7GTP binding assay was performed in EBC-1 and its resistant subline EBC-1PHAR treated with AZD1208 (1 μmol/L) or BEZ235 (0.5 μmol/L) for 5 hours. Elutes and cell lysates were immunoblotted and probed with the indicated antibodies.
Ningfei An et al. Cancer Res 2015;75:
©2015 by American Association for Cancer Research