Figure 6

Translational control during endothelial differentiation and rapamycin treatment. a) Preparation of total RNA and ribosome-enriched RNA. RoSH2 cells, RoSH2 induced to differentiate by plating on matrigel for 48 hours and RoSH2 cells treated with 50 ηM rapamycin for 48 hours were harvested. Total and ribosome-enriched RNAs were quantitated by absorbance at 260 nm. 2 μg of RNA from each of the two cellular fractions were separated on a 1.0% agarose gel, stained with ethidium bromide and visualized under UV illumination. Top panel Representative RNA samples from total and ribosome-enriched RNAs prepared from undifferentiated (Undiff), differentiated (Diff) and rapamycin-treated (Rapa) RoSH2 cells are shown. Bottom panel For each sample, 1 μl of serially diluted RNA sample mixed with 1 μl of 0.5 ηg/μl ethidium bromide was visualized under UV illumination to verify RNA loading in each lane; RNA was isolated from total cellular extract (total) and ribosome-enriched subcellular fraction (ribo) prepared from RoSH2 cells before and 48 hours after induction of endothelial differentiation by plating matrigel-coated plates; b) Distribution of rpL5 mRNA in total and ribosome-enriched RNAs before and after differentiation. RT-PCR using oligo-dT-primed cDNA, and Rpl5 and Fkbp12 specific primers was performed on 10 fold serial dilution i.e. 1×, 10× and 100× of RNAs.; c) RT-PCR analysis of transcript abundance in RoSH2 cells before and after differentiation. RoSH2 cells were induced to undergo endothelial differentiation by plating on matrigel. Total and ribosome-enriched RNA were purified at 0 and 48 hours and analyzed by RT-PCR for transcript abundance of G1 cyclins and endothelial receptors; d) Western blot analysis. RoSH2 cells were induced to undergo endothelial differentiation by plating on matrigel and at 0, 6, 24 and 48 hours, cell lysates were prepared and assayed by western blot analysis. Tsc2 protein was used as an internal control for loading between lanes and between blots; e) Effect of rapamycin on Rpl5, cyclinD2 and Tek transcript abundance in total and ribosome-enriched RNA. Total and ribosome-enriched RNA were isolated from RoSH2 cells treated for 0 and 48 hours with 50 ηM rapamycin treatment and analyzed by RT-PCR; f) Western blot analysis. Cell lysates from rapamycin-treated RoSH2 cells at 0, 6, 24 and 48 hours were assayed by western blot analysis for cyclinD2 and Tek. Tsc2 protein was used as an internal control for loading between lanes and between blots.