Fig 2: Rapamycin inhibits the accumulation of new rRNA and promotes its decay. (A) HeLa cells maintained in medium containing serum were treated with or without rapamycin at the indicated concentrations for 90 min. Levels of phosphorylated and total RpS6 were analyzed by western blot; data are typical of three separate experiments. (B) Uridine uptake was measured after 20 s incubation with [3H]uridine in the absence or presence of 5 nM rapamycin (added 90 min prior to the uptake assay). The radioactivity was determined by scintillation counting, normalized to the total protein level for that sample and plotted as a percentage of the uptake into non-treated HeLa cells. (C and D) HeLa cells were pre-incubated with rapamycin (5 or 100 nM, as indicated) for 15 min before the addition of 4-TU. Total RNA was extracted 30 min or 75 min after addition of 4-TU and processed to measure levels of labeled rRNA species as described in the ‘Materials and Methods’ section. Levels of purified labeled rRNA were compared with those in untreated controls that had been labelled for 45 min, data being normalized to the total level of 18 S rRNA. Data are presented as mean ± SEM (n = 3). The times given below each column indicate the period of 4-TU labeling. (E) HeLa cells were pre-incubated with 4-TU for 4 h. After washing with 1 × PBS to remove excess 4-TU and transfer to fresh medium, rapamycin (100 nM) was added. RNA was extracted and samples were processed as for Panels C and D. Data are presented and expressed as described in the ‘Materials and Methods’ section, in this case being normalized to the 45 min control and given as mean ± SEM (n = 3).

Fig 3: Effect of rapamycin and its removal on ribosome distribution. HeLa cells were incubated with 100 nM rapamycin for 4 h and then the medium was replaced with fresh medium without the inhibitor for 2 h before the lysis. (A) Total RNA was extracted and subjected to northern blot analysis for pre-rRNA species. The membrane was hybridized with probes ITS1, ITS2, 18S and 28S as previously described. (B) Polysomal profile after rapamycin removal of HeLa cells. The polysomal and non-polysomal fractions are indicated. (C) Northern blot analysis of cytoplasmic RNA extracts from each fraction of sucrose gradient and analyzed with indicated probes (RpS6, RpS19 and ß-actin). (D) Quantification of the signals is reported as a linear plot of percentage of mRNAs in each fraction.

Fig 4: Overview of the CD200R signaling pathway.p-Akt is inhibited by Dok2, which also reqruits RasGAP. RasGAP inhibits p-Erk, resulting in inhibition of IL-8. Inhibition of p-rpS6 by CD200R is a result of the inhibition of both p-Akt and p-Erk, although CD200R-mediated p-rpS6 inhibition might also be the result of another yet unknown pathway. This figure was adapted from Van der Vlist et al. [17].