Extended Data Fig. 5: PDCD4 inhibits eIF4F complex formation and cooperates with eFT508 to reduce Hgf, Spp1 and Bgn levels.
a, Western blot showing the protein levels of PTEN, pSer473-AKT, AKT total, pSer235-S6, eIF4E, MNK1 and representative HSP90 in wild-type prostate and Ptenpc−/−;Trp53pc−/− prostate cancer. The experiment was performed once with n = 3 mice for each group. b, Heatmap showing PDCD4 mRNA levels in the indicated genetic background of prostate cancer compared to wild-type prostate (total mRNA expression determined by RNA-seq). c, Western blot showing the levels of Pten and PDCD4 in the indicated settings (top). Cap pull-down assay showing the levels of eIF4G, eIF4A, eIF4E and p-eIF4E in input, cap pull-down and sepharose control beads. Densitometry values of the cap pull-down normalized to the input are indicated for each band (bottom). d, Western blot showing the levels of p-eIF4E and eIF4E after RNA immunoprecipitation with the respective antibody in Pten-sh TC1 prostate cancer cells. e, Western blot showing the levels of HGF, SPP1, BGN, p-eIF4E and representative HSP90 in Pten-sh TC1 cell line upon the indicated concentration of eFT508. f, Western blot showing the levels of HGF, SPP1, BGN, p-eIF4E, PDCD4 and representative HSP90 in Pten-sh TC1 cell line upon 500 nM eFT508 treatment and PDCD4 rescue. g, Polysome profiles of vehicle, 500 nM eFT508-treated, Pdcd4-overexpressing Pten-sh cells and eFT508-treated / Pdcd4-overexpressing Pten-sh cells. h, Distribution of Hgf, Spp1 and Bgn mRNA levels in the fractions derived from the sucrose gradient fractionation in Pten-sh TC1 cells, determined by qRT-PCR (n = 5 independent experiments for Hgf and Spp1; n = 4 independent experiments for Bgn; n = 3 independent experiments for Actinb). The percentages of Hgf, Spp1 and Bgn mRNA distributed in each fraction are shown. v = vehicle; e = eFT508; p = pdcd4; e + p = eFT508 + pdcd4. Data are mean ± SD. Statistical analysis between all groups (ordinary two-way ANOVA followed by Tukey’s multiple comparisons test). i, Western blot showing the levels of PDCD4 and p-eIF4E in human PC3 prostate cancer cell line. j, Translation efficiency (polysomal mRNA expression/ total mRNA expression) of HGF, SPP1, BGN, ISG15 and PDGFB upon 500 nM eFT508 treatment and PDCD4 rescue in human PC3 prostate cancer cell line (n = 3 independent experiments). Data are mean ± SD. Statistical analysis between all groups: (RM one-way ANOVA followed by Tukey’s multiple comparisons test). Densitometry values normalized to the housekeeping are indicated for each band in (a) and (e-f) and (i). The experiment was repeated at least two independent times with similar results in (c-f) and (i).
