Figure 1.

Representative polyribosome profiles obtained from xenografts samples and RT-PCR of polyribosomal RNA. A. Collected xenografts were homogenized and clarified by centrifugation. The resulting extracts were loaded onto a 50% sucrose cushion and centrifuged at 35 k rpm for 2h. The polyribosome-enriched resultant pellet was then resuspended and loaded on a 15%–55% sucrose gradient and the polyribosome profile was obtained by continuous UV (absorbance at 254 nm) monitoring during unloading. RNA was extracted from pooled fractions and verified for quality and integrity by a TapeStation 2200 (Agilent Technologies, Santa Clara, CA, USA), then analyzed by RT-PCR. LP: Light polyribosomes; HP: Heavy polyribosomes. B. Semi-quantitative RT-PCR of purified RNA from LP and HP using specific oligos for either human (h) β-ACTIN or ATF4, mouse (m) β-ACTIN or ATF4, and 28S rRNA. The control oligos, specific to mβ-ACTIN or mATF4, fail to amplify any products, attesting that our polyribosomes preparations are free of murine cross contamination. As expected, the results show that β-ACTIN mRNA is highly enriched in HP as compared to LP indicating efficient translation of this mRNA. On the contrary, the mRNA encoding ATF4 is moderately enriched in HP as compared to LP in keeping with the normal low expression of this regulatory gene, as compared to the high expression of housekeeping genes such as β-ACTIN.