Fig. 5
Knockdown of CSDE1 impairs post-transcriptional regulation of FABP7 and VIM. a Polysome profiles indicate no differences in the ribosome pool upon CSDE1 knockdown (graph is representative of 3 independent experiments). b Western blot analysis with antibodies to CSDE1, OCT4, PAX6, FABP7 and VIM of H9 hESCs daily monitored to remove differentiated cells. β-actin is the loading control. c Graph (relative expression to NT shRNA H9 hESCs) represents the mean ± s.e.m. of three independent experiments with three biological replicates. d Ribonucleoprotein immunoprecipitation (RIP) with CSDE1 antibody. Quantitative PCR analysis of the indicated genes is expressed as fold enrichment over RIP performed with FLAG control antibody. Graph (relative enrichment to FLAG antibody) represents the mean ± s.e.m. (n = 4 independent experiments). e mRNA levels were determined after the indicated time of actinomycin D (ActD) treatment (5 μg ml−1) by qPCR. For each gene, the right graph corresponds to a representative experiment showing the percentage of mRNA relative to time = 0. In the left panel, mRNA degradation is shown as relative slope to non-targeting (NT) shRNA hESCs (mean ± s.e.m. of five independent experiments). The time course experiments with ActD treatment were established for every gene depending on the mRNA degradation rates observed in the NT shRNA samples. f Polysome profiling experiments followed by qPCR analysis. Total and polysome fractions mRNA levels are expressed as relative values to total and polysome fractions of NT shRNA hESCs, respectively. Graph represents the mean ± s.e.m. (n = 4 independent experiments). In c, d and e the statistical comparisons were made by Student’s t-test for unpaired samples. In f the statistical comparisons were made by Student’s t-test for paired samples (the mRNA polysome fraction was paired to its corresponding total mRNA in each independent experiment). P-value: *(P < 0.05), **(P < 0.01), ***(P < 0.001), **** (P < 0.0001)
